Abstract:
The invention relates to a method of diagnosis of Huntington&#39;s Disease in a diagnostic sample of a valid body tissue taken from a human subject, which comprises detecting an altered concentration of a protein in the diagnostic sample, compared with a sample of a control human subject, the protein being selected from: Swiss Prot accession number: Protein name; P10909: Clusterin precursor; P00738: Haptoglobin precursor; P01009: Alpha-1-antitrypsin precursor; P01024: Complement C3 precursor; P01620: 1g kappa chain V-III region; P01834: 1 g kappa chain C region P01842: 1g lambda chain C regions; P01857: 1g gamma-1 chain C region; P01859: Ig gamma-2 chain C region; P01876: 1g alpha-1 chain C region P02647: Apolipoprotein A-I precursor; P02649: Apolipoprotein E precursor; P02652: Apolipoprotein A-II precursor; P02655: Apolipoprotein C-II precursor; P02656: Apolipoprotein C-II precursor P02671: Fibrinogen alpha/alpha-E chain precursor; P02763: Alpha-1-acid glycoprotein 1 precursor; P02766: Transthyretin precursor; P02768: Serum albumin precursor; P02787: Serotransferrin precursor; P04196: Histidine-rich glycoprotein precursor; P06727: Apolipoprotein A-IV precursor; P19652: Alpha-1-acid glycoprotein 2 precursor; P68871/P02042: Hemoglobin beta chain/Hemoglobin delta chain; P60709: Beta actin.

Description:
RELATED APPLICATION INFORMATION 
     This application is a 371 national stage entry of PCT/GB2005/004700, filed Dec. 7, 2005, which claims the benefit of priority from UK application no. 0521762.5, filed on Oct. 25, 2005, and UK application no. 0426859.9, filed on Dec. 7, 2004. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to the diagnosis of neurodegenerative diseases, namely Huntington&#39;s Disease (HD). 
     2. Description of the Related Art 
     Huntington&#39;s disease is autosomal dominantly inherited and is caused by a CAG repeat expansion in the IT15 gene on chromosome 4, resulting in production of a long polyglutamine stretch. The disease is associated with progressive and severe degeneration of the striatum and cortex of the brain, and is clinically characterised by a movement disorder, behavioural problems and dementia. The mean age of onset is 40 years and life expectancy is 15-20 years. 
     The disease is clinically heterogeneous and there are difficulties in the assessment of disease progression in this illness that have led to the need for further methods to be developed to aid the development of therapeutic trials for this disease. 
     SUMMARY OF THE INVENTION 
     The invention provides the use of specified marker proteins and their partners in or for the diagnosis of HD. These marker proteins have been found to be differentially expressed in two dimensional electrophoresis of plasma samples and Surface Enhanced Laser Desorption Ionisation (SELDI) time of flight mass spectrometry profiling experiments. 
     The marker proteins and their differential expression characteristics are as follows:
     1. Protein present in an increased concentration in a HD sample, compared with a control: clusterin precursor (SwissProt Acc. No. P10909);   2. Further proteins present in an increased or decreased concentration in a HD sample, compared with a control, as listed below;   3. Proteins present in an increased concentration in HD samples, compared with a control: beta-actin (SwissProt Acc. No. P60709) and apolipoprotein A-IV precursor (SwissProt Acc. No. P06727).   

     Thus, the invention includes specifically:
     1. A method of diagnosis of Huntington&#39;s Disease, including assessment of disease stage, in a diagnostic sample of a valid body tissue taken from a human subject, which comprises detecting an altered concentration of a protein in the diagnostic sample, compared with a sample of a control human subject, the protein being selected from:   

     
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Swiss Prot 
                   
               
               
                   
                 accession number 
                 Protein name 
               
               
                   
                   
               
             
             
               
                   
                 P10909 
                 Clusterin precursor 
               
               
                   
                 P00738 
                 Haptoglobin precursor 
               
               
                   
                 P01009 
                 Alpha-1-antitrypsin precursor 
               
               
                   
                 P01024 
                 Complement C3 precursor 
               
               
                   
                 P01620 
                 Ig kappa chain V-III region 
               
               
                   
                 P01834 
                 Ig kappa chain C region 
               
               
                   
                 P01842 
                 Ig lambda chain C regions 
               
               
                   
                 P01857 
                 Ig gamma-1 chain C region 
               
               
                   
                 P01859 
                 Ig gamma-2 chain C region 
               
               
                   
                 P01876 
                 Ig alpha-1 chain C region 
               
               
                   
                 P02647 
                 Apolipoprotein A-I precursor 
               
               
                   
                 P02649 
                 Apolipoprotein E precursor 
               
               
                   
                 P02652 
                 Apolipoprotein A-II precursor 
               
               
                   
                 P02655 
                 Apolipoprotein C-II precursor 
               
               
                   
                 P02656 
                 Apolipoprotein C-III precursor 
               
               
                   
                 P02671 
                 Fibrinogen alpha/alpha-E chain precursor 
               
               
                   
                 P02763 
                 Alpha-1-acid glycoprotein 1 precursor 
               
               
                   
                 P02766 
                 Transthyretin precursor 
               
               
                   
                 P02768 
                 Serum albumin precursor 
               
               
                   
                 P02787 
                 Serotransferrin precursor 
               
               
                   
                 P04196 
                 Histidine-rich glycoprotein precursor 
               
               
                   
                 P06727 
                 Apolipoprotein A-IV precursor 
               
               
                   
                 P19652 
                 Alpha-1-acid glycoprotein 2 precursor 
               
               
                   
                 P68871/P02042 
                 Hemoglobin beta chain/Hemoglobin delta chain 
               
               
                   
                 P60709 
                 Beta actin 
               
               
                   
                   
               
             
          
         
       
         
         2. A method as defined in 1 above, which comprises detecting an increased concentration of a protein in the diagnostic sample, compared with a sample of a control human subject, the protein being a clusterin precursor (SwissProt Acc No. P10909). 
         3. A method according to claim  1 , which comprises detecting an increased concentration of a protein in the diagnostic sample, compared with a sample of a control human subject, the protein being: 
         beta actin (SwissProt Acc. No. P60709) or 
         apolipoprotein A-IV precursor (SwissProt Acc. No. P06727). 
       
    
     The marker protein can be present in the body tissue in any biologically relevant form, e.g. in a glycosylated, phosphorylated, multimeric or precursor form. 
     Although there is a high degree of confidence in the identification of the marker proteins specified above, the invention can be defined alternatively in terms of the proteins within the differentially expressed spots on a two dimensional electrophoretic gel, namely those identified in  FIG. 2  herein, without regard to the names and database identifications given above. 
     DEFINITIONS 
     The term “differentially expressed” means that the stained protein-bearing spots are present at a higher or lower optical density in the gel from the sample taken for diagnosis (the “diagnostic sample”) than the gel from a control or other comparative sample. It follows that the proteins are present in the plasma of the diagnostic sample at a higher or lower concentration than in the control or other comparative sample. 
     The term “control” refers to a normal human subject, i.e. one not suffering from a neurodegenerative disease, and also to a sample taken from the same human subject that provided the diagnostic sample, but at an earlier time. 
     The terminology “increased/decreased concentration . . . compared with a sample of a control” does not imply that a step of comparing is actually undertaken, since in many cases it will be obvious to the skilled practitioner that the concentration is abnormally high. Further, when the stages of HD are being monitored progressively, the comparison made can be with the concentration previously seen in the same subject in earlier progression of the disease. 
     The term “binding partner” includes a substance that recognises or has affinity for the marker protein. It may or may not itself be labelled. 
     The term “marker protein” includes all biologically relevant forms of the protein identified. 
     The term “diagnosis”, as used herein, includes determining whether the relevant disease is present or absent and also includes, in relation to Huntington&#39;s Disease, determining the stage to which it has progressed. The diagnosis can serve as the basis of a prognosis as to the future outcome for the patient and for monitoring efficacy of treatment. 
     The term “valid body tissue” means any tissue in which it may reasonably be expected that a marker protein would accumulate in relation to HD. While it will principally be a body fluid, it also includes brain or nerve tissue, it being understood that the diagnosis can be post mortem. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  is a photograph of a typical two dimensional gel performed for analytical purposes, by the method described in Example 1 below. The molecular weight (relative molecular mass) is shown on the ordinate in kiloDaltons. Molecular weight markers are shown at the left-hand side. The isoelectric point (pI) is shown on the ordinate, increasing from left to right. 
         FIG. 2  is similar to  FIG. 1 , but showing spots  1713  and  1960  in a sample derived from an HD patient. 
         FIGS. 3 ,  4  and  5  show box and whisker plots of Western blotting results for a marker for HD, as more fully explained in Example 2. 
         FIG. 6  shows scatter-plots of replicate spectra from the Q10-Tris data set as explained in Example 3. 
         FIG. 7  is a Venn diagram displaying the number and overlap of statistically different peaks in three experimental data sets, as explained in Example 3. 
         FIG. 8  shows box and whisker plots of significantly different peak intensities, as explained in Example 3. 
     
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     A preferred method of diagnosis comprises performing a binding assay for the marker protein. Any reasonably specific binding partner can be used. Preferably the binding partner is labelled. Preferably the assay is an immunoassay, especially between the marker and an antibody that recognises the protein, especially a labelled antibody. It can be an antibody raised against part or all of it, most preferably a monoclonal antibody or a polyclonal anti-human antiserum of high specificity for the marker protein. 
     Thus, the marker proteins described above are useful for the purpose of raising antibodies thereto which can be used to detect the increased or decreased concentration of the marker proteins present in a diagnostic sample. Such antibodies can be raised by any of the methods well known in the immunodiagnostics field. 
     The antibodies may be anti- to any biologically relevant state of the protein. Thus, for example, they could be raised against the unglycosylated form of a protein which exists in the body in a glycosylated form, against a more mature form of a precursor protein, e.g. minus its signal sequence, or against a peptide carrying a relevant epitope of the marker protein. 
     The sample can be taken from any valid body tissue, especially body fluid, of a (human) subject, but preferably blood, plasma or serum. Other usable body fluids include cerebrospinal fluid (CSF), urine and tears. 
     According to another embodiment of the invention, the diagnosis is carried out post mortem on a body tissue of neurological origin relevant to HD, such as from the brain or nerves. The tissue is pre-treated to extract proteins therefrom, including those that would be present in the blood of the deceased, so as to ensure that the relevant marker proteins specified above will be present in a positive sample. For the purposes of this patent specification, such an extract is equivalent to a body fluid. 
     By way of example, brain tissue is dissected and sub-sections solubilised in 2-D gel lysis buffer (e.g. as described below), in a ratio of about 100 mg tissue to 1 ml buffer. 
     The preferred immunoassay is carried out by measuring the extent of the protein/antibody interaction. Any known method of immunoassay may be used. A sandwich assay is preferred. In this method, a first antibody to the marker protein is bound to the solid phase such as a well of a plastics microtitre plate, and incubated with the sample and with a labelled second antibody specific to the protein to be assayed. Alternatively, an antibody capture assay could be used. Here, the test sample is allowed to bind to a solid phase, and the anti-marker protein antibody is then added and allowed to bind. After washing away unbound material, the amount of antibody bound to the solid phase is determined using a labelled second antibody, anti- to the first. 
     In another embodiment, a competition assay is performed between the sample and a labelled marker protein or a peptide derived therefrom, these two antigens being in competition for a limited amount of anti-marker protein antibody bound to a solid support. The labelled marker protein or peptide thereof could be pre-incubated with the antibody on the solid phase, whereby the marker protein in the sample displaces part of the marker protein or peptide thereof bound to the antibody. 
     In yet another embodiment, the two antigens are allowed to compete in a single co-incubation with the antibody. After removal of unbound antigen from the support by washing, the amount of label attached to the support is determined and the amount of protein in the sample is measured by reference to standard titration curves established previously. 
     The label is preferably an enzyme. The substrate for the enzyme may be, for example, colour-forming, fluorescent or chemiluminescent. 
     The binding partner in the binding assay is preferably a labelled specific binding partner, but not necessarily an antibody. For example, when the marker protein is alpha-1-antitrypsin, the specific binding partner can be trypsin. The binding partner will usually be labelled itself, but alternatively it may be detected by a secondary reaction in which a signal is generated, e.g. from another labelled substance. 
     It is highly preferable to use an amplified form of assay, whereby an enhanced “signal” is produced from a relatively low level of protein to be detected. One particular form of amplified immunoassay is enhanced chemiluminescent assay. Conveniently, the antibody is labelled with horseradish peroxidase, which participates in a chemiluminescent reaction with luminol, a peroxide substrate and a compound which enhances the intensity and duration of the emitted light, typically 4-iodophenol or 4-hydroxycinnamic acid. 
     Another preferred form of amplified immunoassay is immuno-PCR. In this technique, the antibody is covalently linked to a molecule of arbitrary DNA comprising PCR primers, whereby the DNA with the antibody attached to it is amplified by the polymerase chain reaction. See E. R. Hendrickson et al., Nucleic Acids Research 23: 522-529 (1995). The signal is read out as before. 
     Alternatively, the diagnostic sample can be subjected to two dimensional gel electrophoresis to yield a stained gel and the increased or decreased concentration of the protein detected by an increased an increased or decreased intensity of a protein-containing spot on the stained gel, compared with a corresponding control or comparative gel. The relevant spots, diseases identified and differential expression are those listed in Table 1 below. The invention includes such a method, independently of the marker protein identification given above and in Table 2. 
     The diagnosis does not necessarily require a step of comparison of the concentration of the protein with a control, but it can be carried out with reference either to a control or a comparative sample. Thus, in relation to Huntington&#39;s disease the invention can be used to determine the stage of progression, if desired with reference to results obtained earlier from the same patient or by reference to standard values that are considered typical of the stage of the disease. In this way, the invention can be used to determine whether, for example after treatment of the patient with a drug or candidate drug, the disease has progressed or not. The result can lead to a prognosis of the outcome of the disease. 
     The invention further includes the use for a diagnostic (and thus possibly prognostic) or therapeutic purpose of a partner material which recognises, binds to or has affinity for a marker protein specified above and/or represented by a differentially expressed two dimensional gel electrophoretic spot shown in  FIG. 2  herein. Thus, for example, antibodies to the marker proteins, appropriately humanised where necessary, may be used to treat HD. The partner material will usually be an antibody and used in any assay-compatible format, conveniently an immobilised format, e.g. as beads or a chip. Either the partner material will be labelled or it will be capable of interacting with a label. 
     The invention further includes a kit for use in a method of diagnosis, which comprises a partner material, as described above, in an assay-compatible format, as described above, for interaction with a protein present in the diagnostic sample. 
     The diagnosis can be based on the differential expression of one, two, three or more of the marker proteins. Further, it can be part of a wider diagnosis in which two or more different diseases are diagnosed. Both vCJD and Huntington&#39;s can be diagnosed together and either or both of those along with at least one other disease, which may or may not be neurological, in the same sample of body fluid, by a method which includes detecting an increased concentration of another protein in the diagnostic sample, compared with a sample of a control, normal human subject. These other disease(s) can be any which are diagnosable in a body fluid. They may be neurological, e.g. another transmissible spongiform encephalopathy, Parkinson&#39;s Disease, meningitis, but are not necessarily neurological, for example toxic shock syndrome, MRSA or Celiac disease. 
     Thus, in particular, it is contemplated within the invention to use an antibody chip or array of chips, capable of diagnosing one or more proteins that interact with that antibody. 
     The following Examples illustrate the invention. 
     EXAMPLE 1 
     Ten plasma samples were taken from patients (4 female, 6 male) who were diagnosed with variant CJD (vCJD) serving as a neurological disease control, ten from patients (7 female, 3 male) diagnosed by genetic testing as having Huntington&#39;s Disease (HD) and ten from controls, i.e. normal patients (8 female, 2 male) not having any neuropathological symptoms. 
     Albumin and IgG were removed from the samples using a kit supplied by Amersham Biosciences UK Ltd. This kit contains an affinity resin containing antibody that specifically removes albumin and IgG directly from whole human serum and plasma samples. It is claimed that more than 95% albumin and more than 90% IgG removal from 15 μl human serum/plasma can be achieved, thereby increasing the resolution of lower abundance proteins in subsequent electrophoresis. A microspin column is used, through which the unbound protein is eluted. 
     Depletion was carried out according to the manufacturer&#39;s instructions using a starting volume of 15 μl of crude plasma sample. The resin was added to the plasma, the mixture incubated with shaking, transferred to a microspin column, centrifuged and the filtrate collected. The resulting depleted sample was concentrated and de-salted by acetone precipitation (as recommended in the instructions of the kit). The acetone was decanted and the pellets were re-suspended in standard 2-D gel lysis buffer (9.5 M urea, 2% CHAPS, 1% DTT, 0.8% Pharmalyte, pH 3-10, protease inhibitors (1 tablet/10 ml lysis buffer) (Roche). This suspension was used for the two dimensional gel electrophoresis. 
     Since the depletion kit does not provide the user with a protocol to “strip off” the proteins bound to the column, a standard chromatography method was adopted for doing this, which is to use a 0.1 M Glycine-HCl, pH 2.5 buffer. All corresponding bound fractions were stored at −80° C. for later use in another experiment. 
     Two dimensional gel electrophoresis was performed according to J. Weekes et al., Electrophoresis 20: 898-906 (1999) and M. Y. Heinke et al., Electrophoresis 20: 2086-2093 (1999), using 18 cm immobilised pH 3-10 non-linear gradient strips (IPGs). The second dimension was performed using 12% T SDS polyacrylamide gel electrophoresis. For the initial analysis, the gels were loaded with 75 micrograms of protein. The gels were silver-stained with the analytical OWL silver stain (Insight Biotechnologies, UK). 
     Quantitative and qualitative image analysis was performed using the software Progenesis™ Workstation, version 2003.02 (Nonlinear Dynamics Ltd.). The images were processed through the automatic wizard for spot detection, warping and matching. Thereafter, all images underwent extensive manual editing and optimal matching to the reference gel (&gt;80% per gel). Following background subtraction and normalisation to total spot volume, protein spot data was exported to Excel for quantitative statistical analysis and comparisons of qualitative changes. 
     The student t-test, at the 95% confidence interval, was performed for every protein spot that could be compared between the samples from the diseased patients and the controls and which was present in at least 60% of the gels of each group, i.e. at least 6. A log transformation was performed, since this gave a more normal distribution, thus better meeting the assumptions of this test as applied to independent samples. 
     The spots for which a significant increase or decrease was observed in comparisons between the three groups are shown in  FIG. 2  and listed in Table 1. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   
                   
                 Quantitative change 
                   
               
               
                   
                   
                   
                 (Increase/Decrease in intensity 
               
               
                   
                 Spot 
                   
                 of spot in comparisons between 
                 p value (t- 
               
               
                   
                 No. 
                 FIG. 
                 vCJD, HD and control samples). 
                 test) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 1713 
                 2 
                 Inc. vCJD vs. Control 
                 0.003 
               
               
                   
                 1713 
                 5 
                 Inc. HD vs. Control 
                 0.000065 
               
               
                   
                 1960 
                 5 
                 Inc. HD vs. Control 
                 0.004 
               
               
                   
                   
               
             
          
         
       
     
     It will be seen that spot  1713  is one to which particularly high confidence in the results can be attached in relation to the increase in its intensity in the HD samples versus controls. 
     For preparative purposes, further two dimensional gels were then made by the same method, by pooling all samples within each experimental group and loading the gels with 400 micrograms of protein. There were thus three gels prepared, one for each group, which were silver stained, using PlusOne silver stain (Amersham Pharmacia Biosciences UK Ltd.). 
     Normally, the spots were excised from the preparative gels in which they were elevated in intensity, but where this was not possible, they were excised from another gel. After in-gel reduction, alkylation and digestion of the excised material with trypsin, the peptides produced were extracted and subsequently analysed by LC/MS/MS. This procedure involves separation of the peptides by reversed phase HPLC, followed by electrospraying to ionise the sample, as it enters a tandem mass spectrometer. The mass spectrometer records the mass to charge ratio of the peptide precursor ions, which are then individually selected for fragmentation via collisionally induced dissociation (CID). This so-called MS/MS scan allows for the sequence of the peptide to be determined. For each sample, therefore, the data set includes accurately determined molecular weights for multiple peptides present, accompanied by corresponding sequence information. This is then used to identify the protein by searching databases. In the present case, the Mascot search algorithm was used against the National Center for Biotechnology Information (NCBI) non-redundant protein (nr) and SWISS-PROT databases. 
     The results of the identification are shown in Table 2. All the spots of Table 1 that were differentially expressed on the gel were identified as known proteins. The Table shows the geninfo (gi) numbers of the NCBI database and SwissProt Accession numbers. 
     In some instances more than one protein was identified, which signifies that the spot excised contained a mixture of proteins, at least one of which was differentially expressed on the gel. The proteins identified in the database had different molecular weights and isoelectric points, lower or higher, from those evident on the gel. This is entirely usual and can be accounted for by the protein within the gel spot having undergone enzymatic or chemical cleavage or by having been post-translationally modified such as by glycosylation, phosphorylation or the addition of lipids. 
     
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 MW 
                   
                   
                   
                   
               
               
                   
                 (Da) 
                 pI 
                   
                 NCBI nr and 
                 No. peptides 
               
               
                 Spot 
                 from 
                 from 
                 Human 
                 SwissProt 
                 matched (% 
               
               
                 No. 
                 gel 
                 gel 
                 protein identified 
                 Acc. No. 
                 coverage) 
               
               
                   
               
             
             
               
                 1713 
                 43108 
                 5.19 
                 Beta actin 
                 gi/4501885 
                 14 (47%) 
               
               
                   
                   
                   
                   
                 P60709 
               
               
                   
                   
                   
                 Apolipoprotein 
                 gi/4502151 
                  7 (26%) 
               
               
                   
                   
                   
                 A-IV precursor 
                 P06727 
               
               
                 1960 
                 33348 
                 4.77 
                 Clusterin 
                 gi/116533 
                  8 (19%) 
               
               
                   
                   
                   
                   
                 P10909 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 2 
     The following Western blotting experiments were performed to show the use of the invention for monitoring the progression of Huntington&#39;s Disease. 
     Plasma samples were obtained, with appropriate consents, from 55 patients having various stages of Huntington&#39;s Disease and from 15 normal patients, as controls. The experimental groups were: control, pre-symptomatic (PST or P), early (E), moderate (M), 15 samples each and advanced (A), 10 samples. The samples were diluted 1 in 300 with sterile PBS (Sigma) and the protein concentration determined in triplicate, using BSA as a standard and the DC protein assay kit (Bio-Rad Laboratories Ltd, Herts, UK). Master mixes of plasma proteins were subsequently prepared to limit pipetting error and freeze-thawing and to enable identical samples to be run on a number of gels. 
     The samples were denatured at 95° C. for 10 min in Laemmli sample buffer (Sigma) and size-separated using 20 cm×10 cm 12% or 16% Tris-Glycine acrylamide gels (Gel tank: Sci-Plas, Southam, UK). Plasma samples were loaded in groups of 2-4 (see Table 3) to distribute samples over the gel and to limit differences in gel running and transfer efficiency. Proteins were transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech Ltd, Buckinghamshire, UK) for 30 min at 25 volts using a semi-dry blotting apparatus, Trans-Blot SD (Bio-Rad Laboratories Ltd). 
     
       
         
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Gel 1 
                 Gel 2 
               
             
          
           
               
                   
                 HD 
                   
                   
                 HD 
                   
               
               
                   
                 Disease 
                   
                   
                 Disease 
                 Sample 
               
               
                 Well 
                 Stage 
                 Sample No 
                 Well 
                 Stage 
                 No 
               
               
                   
               
             
          
           
               
                 1 
                   
                 Markers 
                 1 
                   
                 Markers 
               
               
                 2 
                 Control 
                 1 
                 2 
                 Control 
                 9 
               
               
                 3 
                 Control 
                 2 
                 3 
                 Control 
                 10 
               
               
                 4 
                 Control 
                 3 
                 4 
                 Control 
                 11 
               
               
                 5 
                 PST 
                 16 
                 5 
                 PST 
                 23 
               
               
                 6 
                 PST 
                 17 
                 6 
                 PST 
                 24 
               
               
                 7 
                 PST 
                 18 
                 7 
                 PST 
                 25 
               
               
                 8 
                 Early 
                 31 
                 8 
                 PST 
                 26 
               
               
                 9 
                 Early 
                 32 
                 9 
                 Early 
                 38 
               
               
                 10 
                 Early 
                 33 
                 10 
                 Early 
                 39 
               
               
                 11 
                 Mod 
                 46 
                 11 
                 Early 
                 40 
               
               
                 12 
                 Mod 
                 47 
                 12 
                 Early 
                 41 
               
               
                 13 
                 Mod 
                 48 
                 13 
                 Mod 
                 54 
               
               
                 14 
                 Mod 
                 49 
                 14 
                 Mod 
                 55 
               
               
                 15 
                 ADV 
                 61 
                 15 
                 Mod 
                 56 
               
               
                 16 
                 ADV 
                 62 
                 16 
                 Mod 
                 57 
               
               
                 17 
                 ADV 
                 63 
                 17 
                 ADV 
                 66 
               
               
                 18 
                 Control 
                 4 
                 18 
                 ADV 
                 67 
               
               
                 19 
                 Control 
                 5 
                 19 
                 ADV 
                 68 
               
               
                 20 
                 PST 
                 19 
                 20 
                 Control 
                 12 
               
               
                 21 
                 PST 
                 20 
                 21 
                 Control 
                 13 
               
               
                 22 
                 PST 
                 21 
                 22 
                 PST 
                 27 
               
               
                 23 
                 PST 
                 22 
                 23 
                 PST 
                 28 
               
               
                 24 
                 Early 
                 34 
                 24 
                 PST 
                 29 
               
               
                 25 
                 Early 
                 35 
                 25 
                 PST 
                 30 
               
               
                 26 
                 Early 
                 36 
                 26 
                 Early 
                 42 
               
               
                 27 
                 Early 
                 37 
                 27 
                 Early 
                 43 
               
               
                 28 
                 Mod 
                 50 
                 28 
                 Early 
                 44 
               
               
                 29 
                 Mod 
                 51 
                 29 
                 Early 
                 45 
               
               
                 30 
                 Mod 
                 52 
                 30 
                 Mod 
                 58 
               
               
                 31 
                 Mod 
                 53 
                 31 
                 Mod 
                 59 
               
               
                 32 
                 ADV 
                 64 
                 32 
                 Mod 
                 60 
               
               
                 33 
                 ADV 
                 65 
                 33 
                 ADV 
                 69 
               
               
                 34 
                 Control 
                 6 
                 34 
                 ADV 
                 70 
               
               
                 35 
                 Control 
                 7 
                 35 
                 Control 
                 14 
               
               
                 36 
                 Control 
                 8 
                 36 
                 Control 
                 15 
               
               
                   
               
             
          
         
       
     
     The transfer efficiency and equal loading of protein samples was assessed by incubating membranes with Ponceau red solution (Sigma). 
     After transfer, membranes were washed with PBS-T (PBS, 0.1% Tween-20, Sigma), incubated (overnight, 4° C.) in blocking buffer (PBS-T, 5% Marvel) and subsequently incubated (2 h, room temperature) with the required primary antibody (see Table 4). After incubation with the primary antibody, membranes were further incubated (1 h, room temperature, 1 in 5000 dilution) with a horseradish peroxidase conjugated sheep anti-mouse (Clusterin and beta-actin, Amersham Pharmacia Biotech Ltd) or rabbit anti-goat secondary antibody (Jackson laboratories, Maine, USA). Thereafter, membranes were washed in PBS-T (6×15 min), incubated with the enhanced chemiluminescent assay reagent ECL-plus (Amersham Pharmacia Biotech Ltd) and the luminescent signal of the protein bands visualised using a Storm 860 scanner (Amersham Pharmacia Biotech Ltd). 
                                                           TABLE 4                   Protein   Acryl-                   conc.   amide       Antibody           (micro-   %       dilution       Protein   grams)   in gel   Antibody   v/v                                Clusterin   2.5   16   Upstate anti-Clusterin   1 in 10,000       precursor           (beta chain)                   (Cat No: 05-354)       Apolipo-   5   12   C20, Santa Cruz   1 in 1,000       protein A-IV           anti-Apolipo-protein A-       precursor           IV (recognising C- and                   N-terminal regions) (Cat                   No: SC-19038)       Beta-actin   300   12   Sigma-Clone AC-74 (Cat   1 in 250                   No: A5316)                    
Data and Statistical Analysis
 
     Boxes of equal size were drawn around each band on Western blot images using ImageQuant (Amersham Pharmacia Biotech Ltd). The volume of all the pixels in each box was calculated, the background value subtracted and the remaining value anlaysed statistically, using the appropriate tests (Table 5). The Levene value (which tests whether the samples have equal variance) was determined for each group of data. If the Levene value was below 0.05 (samples have unequal variance), then the Welch statistic was checked and the Tamhane post hoc test was used. If the Levene value was above 0.05 then ANOVA was used with the Tukey HSD (Honestly Significant Difference) post hoc test. 
     After applying the appropriate post hoc test, a probability value (P) was obtained, less than 0.05 being considered significant. 
     It will be seen that a substantial number of significant or near-significant results (asterisked) at the P&lt;0.05 level were obtained, including many between the moderate group and the control group and between the moderate group and the pre-symptomatic group. 
     The results for one particular day were further analysed by box and whisker plots, for Gel 1 (35 results), Gel 2 (35 results) and Gels 1 &amp; 2 (all 70 results). See  FIGS. 3 to 5 , where C=control, P=pre-symptomatic, E=early, M=moderate and A=advanced HD. The boxes represent the upper and lower quartiles above the median, denoted by the thick line, while the whiskers extend to the observations which are 1.5 times or less than the interquartile distance from the box. Outlier values, more than 1.5× and up to 3× the interquartile range, are shown as a circle, extreme cases, more than 3×, by an asterisk. The outliers and extreme cases were included in the statistical data analysis in Table 5. It will be seen that there was a substantial correlation between stage of the disease up to moderate and the density of the Clusterin precursor band on the gel. 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Statistical analysis of Clusterin precursor Western blots. 
               
             
          
           
               
                 Blot date 
                 Gels 
                 Levene 
                 ANOVA 
                 Welch 
                 Post hoc 
                 Group 
                 P 
               
               
                   
               
             
          
           
               
                 31 
                 1 &amp; 2 
                 0.547 
                 0.047 
                 0.014 
                 Tukey HSD 
                 A &gt; C 
                 0.029 
               
               
                 Aug 
               
               
                 06 
                 1 &amp; 2 
                 0.002 
                 0.075 
                 0.006 
                 Tamhane 
                 M &gt; P 
                 0.041 
               
               
                 Sep 
                   
                   
                   
                   
                   
                 E &gt; P 
                 0.03 
               
               
                 08 
                 1 &amp; 2 
                 0.011 
                 0.001 
                 0.001 
                 Tamhane 
                 M &gt; C 
                 0.003 
               
               
                 Sep 
                   
                   
                   
                   
                   
                 M &gt; P 
                 0.002 
               
               
                   
                   
                   
                   
                   
                   
                 E &gt; C 
                 0.064* 
               
               
                 06 
                 1 
                 0.069 
                 0.004 
                 0.023 
                 Tukey 
                 E &gt; C 
                 0.005 
               
               
                 Sep 
                   
                   
                   
                   
                 HSD 
                 M &gt; C 
                 0.011 
               
               
                 07 
                 1 
                 0.019 
                 0.004 
                 0.002 
                 Tamhane 
                 A &gt; P 
                 0.042 
               
               
                 Sep 
                   
                   
                   
                   
                   
                 M &gt; P 
                 0.004 
               
               
                   
                   
                   
                   
                   
                   
                 A &gt; C 
                 0.055* 
               
               
                 08 
                 1 
                 0.00 
                 0.029 
                 0.013 
                 Tamhane 
                 M &gt; P 
                 0.013 
               
               
                 Sep 
                   
                   
                   
                   
                   
                 E &gt; P 
                 0.089 
               
               
                 06 
                 2 
                 0.028 
                 0.106 
                 0.024 
                 Tamhane 
                 A &gt; P 
                 0.089* 
               
               
                 Sep 
               
               
                 08 
                 2 
                 0.766 
                 0.044 
                 0.051 
                 Tukey 
                 M &gt; C 
                 0.045 
               
               
                 Sep 
                   
                   
                   
                   
                 HSD 
               
               
                   
               
               
                 *= nearly significant at P &lt; 0.05. 
               
             
          
         
       
     
     Apolipoprotein A4 precursor was found to be significantly increased in moderate HD samples when compared to controls in one gel out of six (n=3, gel 1 and gel 2 experiments). 
     Beta-actin: the preliminary Western blots suggest that beta-actin is the protein that is changing in the 2D gel spot  1713 . However, the blots had an extremely high background which inhibited quantification. 
     EXAMPLE 3 
     Introduction 
     Components within the plasma from patients with Huntingdon&#39;s disease (HD) and healthy controls (CON; not age-sex matched) were profiled using surface enhanced laser desorption/ionisation time-of flight mass spectrometry (SELDI). Three experiments were performed, each involving the same set of plasma samples but differing in the chip or wash buffer used. The HD group was further sub-divided into pre—(PRE), early—(EAR), moderate—(MOD) or advanced-disease (ADV). The control and disease groups all consisted of 15 patients samples except for the ADV group, which contained 10 samples. The protein profiles of plasma were obtained using Protein Chips (Ciphergen Biosystems) with either a strong anion exchange surface (SAX, Q10) or a weak cation exchange surface (WCX, CM10). The CM10 chips were equilibrated and washed in only one type of buffer whilst the Q10 chips were analysed following treatment with two alternative buffers. The experiment using Q10 chips washed in 100 mM Tris HCl (pH 9.0) is referred to as “Q10-Tris”. The experiment involving Q10 chips washed in 50 mM sodium acetate (pH 6.5) is referred to as Q10-NaAc. The experiment involving CM10 chips washed in 50 mM ammonium acetate (pH 7.5) is referred to as CM10-AmAc. 
     Data Preparation 
     Calibration: The SELDI-TOF mass spectrometer was calibrated using a mixture of adrenocorticotropic hormone residues 18-39 (ACTH), cytochrome C, myoglobin and bovine serum albumin (BSA). Following acquisition of spectra for the protein profiling experiments, one spectrum was chosen as a reference spectrum (EAR sample 8117 in spot position E) and the corresponding spot over-layered with 1 μL of an aqueous solution containing the calibrant molecules. A further 1 μL of a 20 mg/mL solution of sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) matrix in 50% aqueous acetonitrile with 0.1% trifluoroacetic acid was added to the spot and allowed to dry for approximately 10 min. Spectra were acquired using the settings applied to the original samples and used to create calibration equations that were applied to the spectra, including the reference spectrum. The ions used to calibrate spectra were: singly-charged ACTH, m/z=2,466.72; doubly-charged cytochrome C, m/z=6,181.05; doubly-charged myoglobin, m/z=8,476.78; singly-charged cytochrome C, m/z=12,361.10; singly-charged myoglobin, m/z=16,952.56; doubly-charged BSA, m/z=33,216.00; singly-charged BSA, m/z=66,560.00). In call cases, average m/z values were used because the mass spectrometer was not able to resolve individual isotopic species. Separate calibration equations were produced for the low (2,467-16,952) and high (16,952-66,560) m/z regions of the spectra and the m/z values of peaks in the spectra were assigned using the m/z values from the reference spectrum, calibrated in the appropriate m/z range. Masses referred to in the report are those derived from the calibrated reference spectra. The 95% confidence intervals (CI) of the average masses for the entire set of clinical samples are also given in Table 9. The 95% CI ranges of m/z values were estimated as the mean m/z value of all the matched peaks±two standard deviations. This range has a 95% probability of encompassing the true population mean m/z value and is a valid method of estimation due to the large (&gt;100) number of samples used to derive the parameters of mean and standard deviation. 
     Peak marking: Peaks were manually marked using the tools provided by the ProteinChip software (Ciphergen Biosystems). Prior to peak marking, a baseline subtraction was performed using a fitted peak width of 5-times the expected peak width. For the Q10-Tris data set, a total of 71 peaks were marked across the m/z range 2,505-66,544. For the CM10-AmAc data set, 67 peaks were detected in the m/z range 2,509-65,587. For the Q10-NaAc data set, there were 66 peaks marked in the region 2,628-66,703. Following peak marking, a visual inspection of all spectra was made and the peak intensity data exported to Excel (Microsoft). The masses of matched peaks were checked in Excel and found to all have coefficients of variation of less than 0.90%. There were a small number of missing values in the data sets where peaks failed to be marked. These values were not converted to zeros but instead left as missing values. 
     Pre-processing: Quantile normalisation was performed according to the method of Bolstad et al. (2003) using a script written in the R statistical programming language (www.r-project.org). Prior to normalisation missing values were replaced with the mean peak intensity for spectra in the same group to provide a place-holder during the normalisation. Following normalisation, the place-holder values were converted back to missing values. Peak intensity data for peaks displaying positively-skewed distributions (skew&gt;0.7) were log 10  transformed prior to all data analysis. 
     Correlation Analysis 
     Pearson correlation coefficients were computed for replicate spectra. In the Q10-Tris data set, many of the samples were analysed in duplicate but some were analysed three times and some only once. Where duplicates existed, the correlation coefficient was computed for the pair. Where triplicates existed, three pair-wise correlation coefficients were computed. Where singlets existed, the mean correlation coefficient of that spectrum compared to all spectra was computed from the correlation matrix generated in the R environment. For the remaining data sets (CM10-AmAc and Q10-NaAc), the samples were analysed in duplicate and correlation coefficients were computed only for duplicate spectra. Prior to computing the correlation coefficients, the data were log 10  transformed. This was done because there were many more peaks of low intensity than there were peaks of high intensity, so the correlation is more representative of the relationship between pairs of spectra after log transformation. The correlation data are shown in Table 6. 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Pearson Correlation of replicate samples in the Q10-Tris data set 
               
             
          
           
               
                 Group 
                 Sample 
                 Spot 
                 Chip 
                 Group 
                 Sample 
                 Spot 
                 Chip 
                 Replication 
                 Correlation 
               
               
                   
               
             
          
           
               
                 EAR 
                 13342 
                 C 
                 5000 
                 EAR 
                 13342 
                 G 
                 5008 
                 Duplet 
                 0.98 
               
               
                 MOD 
                 10945 
                 A 
                 5008 
                 MOD 
                 10945 
                 C 
                 5011 
                 Duplet 
                 0.98 
               
               
                 EAR 
                 11262 
                 B 
                 5011 
                 EAR 
                 11262 
                 B 
                 5014 
                 Triplet 
                 0.97 
               
               
                 CON 
                 10653 
                 A 
                 5001 
                 CON 
                 10653 
                 E 
                 5015 
                 Duplet 
                 0.97 
               
               
                 EAR 
                 8117 
                 A 
                 5005 
                 EAR 
                 8117 
                 E 
                 5016 
                 Duplet 
                 0.97 
               
               
                 EAR 
                 8206 
                 A 
                 5003 
                 EAR 
                 8206 
                 H 
                 4998 
                 Duplet 
                 0.97 
               
               
                 MOD 
                 8131 
                 E 
                 5010 
                 MOD 
                 8131 
                 E 
                 5003 
                 Duplet 
                 0.97 
               
               
                 MOD 
                 8126 
                 C 
                 5017 
                 MOD 
                 8126 
                 H 
                 5005 
                 Duplet 
                 0.97 
               
               
                 EAR 
                 12112 
                 A 
                 5015 
                 EAR 
                 12112 
                 F 
                 4999 
                 Duplet 
                 0.97 
               
               
                 MOD 
                 13165 
                 B 
                 5005 
                 MOD 
                 13165 
                 C 
                 5001 
                 Duplet 
                 0.97 
               
               
                 CON 
                 8413 
                 A 
                 5016 
                 CON 
                 8413 
                 E 
                 5002 
                 Duplet 
                 0.97 
               
               
                 EAR 
                 11262 
                 B 
                 5011 
                 EAR 
                 11262 
                 B 
                 5013 
                 Triplet 
                 0.97 
               
               
                 EAR 
                 10837 
                 B 
                 5001 
                 EAR 
                 10837 
                 H 
                 5001 
                 Duplet 
                 0.96 
               
               
                 ADV 
                 13272 
                 E 
                 5005 
                 ADV 
                 13272 
                 F 
                 4998 
                 Duplet 
                 0.96 
               
               
                 CON 
                 11207 
                 G 
                 5011 
                 CON 
                 11207 
                 G 
                 5007 
                 Duplet 
                 0.96 
               
               
                 EAR 
                 11298 
                 H 
                 5014 
                 EAR 
                 11298 
                 D 
                 5003 
                 Triplet 
                 0.96 
               
               
                 CON 
                 8358 
                 A 
                 5007 
                 CON 
                 8358 
                 C 
                 5006 
                 Duplet 
                 0.96 
               
               
                 CON 
                 10841 
                 B 
                 5000 
                 CON 
                 10841 
                 F 
                 5008 
                 Duplet 
                 0.96 
               
               
                 CON 
                 8114 
                 C 
                 5018 
                 CON 
                 8114 
                 E 
                 4999 
                 Duplet 
                 0.96 
               
               
                 EAR 
                 8355 
                 B 
                 4998 
                 EAR 
                 8355 
                 H 
                 5007 
                 Duplet 
                 0.96 
               
               
                 ADV 
                 13164 
                 F 
                 5011 
                 ADV 
                 13164 
                 F 
                 5001 
                 Duplet 
                 0.96 
               
               
                 CON 
                 10947 
                 C 
                 5003 
                 CON 
                 10947 
                 G 
                 5017 
                 Duplet 
                 0.96 
               
               
                 MOD 
                 10866 
                 A 
                 4999 
                 MOD 
                 10866 
                 A 
                 5012 
                 Duplet 
                 0.96 
               
               
                 MOD 
                 10843 
                 C 
                 5007 
                 MOD 
                 10843 
                 D 
                 5000 
                 Duplet 
                 0.96 
               
               
                 PRE 
                 12323 
                 D 
                 5004 
                 PRE 
                 12323 
                 F 
                 5018 
                 Duplet 
                 0.96 
               
               
                 MOD 
                 10868 
                 C 
                 5004 
                 MOD 
                 10868 
                 G 
                 4999 
                 Duplet 
                 0.96 
               
               
                 ADV 
                 11841 
                 F 
                 5007 
                 ADV 
                 11841 
                 H 
                 5006 
                 Duplet 
                 0.96 
               
               
                 MOD 
                 8119 
                 B 
                 5015 
                 MOD 
                 8119 
                 H 
                 5008 
                 Duplet 
                 0.96 
               
               
                 PRE 
                 12575 
                 E 
                 5000 
                 PRE 
                 12575 
                 F 
                 5010 
                 Duplet 
                 0.96 
               
               
                 EAR 
                 11262 
                 B 
                 5013 
                 EAR 
                 11262 
                 B 
                 5014 
                 Triplet 
                 0.96 
               
               
                 EAR 
                 11289 
                 D 
                 5006 
                 EAR 
                 11289 
                 H 
                 5011 
                 Duplet 
                 0.96 
               
               
                 MOD 
                 12492 
                 E 
                 5018 
                 MOD 
                 12492 
                 G 
                 5002 
                 Duplet 
                 0.96 
               
               
                 EAR 
                 11298 
                 H 
                 5014 
                 EAR 
                 11298 
                 H 
                 5013 
                 Triplet 
                 0.95 
               
               
                 MOD 
                 8125 
                 A 
                 5018 
                 MOD 
                 8125 
                 F 
                 5016 
                 Duplet 
                 0.95 
               
               
                 PRE 
                 12581 
                 C 
                 5015 
                 PRE 
                 12581 
                 C 
                 5005 
                 Duplet 
                 0.95 
               
               
                 PRE 
                 11260 
                 B 
                 4999 
                 PRE 
                 11260 
                 B 
                 5002 
                 Duplet 
                 0.95 
               
               
                 ADV 
                 8113 
                 B 
                 5006 
                 ADV 
                 8113 
                 D 
                 5002 
                 Duplet 
                 0.95 
               
               
                 EAR 
                 8116 
                 B 
                 5017 
                 EAR 
                 8116 
                 F 
                 5012 
                 Duplet 
                 0.95 
               
               
                 ADV 
                 8201 
                 H 
                 5010 
                 ADV 
                 8201 
                 H 
                 4997 
                 Duplet 
                 0.95 
               
               
                 PRE 
                 12360 
                 B 
                 5008 
                 PRE 
                 12360 
                 H 
                 4999 
                 Duplet 
                 0.95 
               
               
                 CON 
                 10969 
                 A 
                 5017 
                 CON 
                 10969 
                 H 
                 5004 
                 Duplet 
                 0.94 
               
               
                 ADV 
                 8391 
                 D 
                 5012 
                 ADV 
                 8391 
                 D 
                 4999 
                 Duplet 
                 0.94 
               
               
                 MOD 
                 8144 
                 B 
                 5016 
                 MOD 
                 8144 
                 G 
                 5012 
                 Duplet 
                 0.94 
               
               
                 CON 
                 13166 
                 A 
                 5011 
                 CON 
                 13166 
                 E 
                 5012 
                 Duplet 
                 0.94 
               
               
                 CON 
                 8421 
                 G 
                 5014 
                 CON 
                 8421 
                 G 
                 4998 
                 Triplet 
                 0.94 
               
               
                 EAR 
                 11205 
                 D 
                 5010 
                 EAR 
                 11205 
                 H 
                 5017 
                 Duplet 
                 0.94 
               
               
                 PRE 
                 12127 
                 D 
                 5007 
                 PRE 
                 12127 
                 H 
                 5015 
                 Duplet 
                 0.94 
               
               
                 PRE 
                 13262 
                 G 
                 5016 
                 PRE 
                 13262 
                 H 
                 5012 
                 Duplet 
                 0.94 
               
               
                 EAR 
                 12363 
                 D 
                 5018 
                 EAR 
                 12363 
                 F 
                 5002 
                 Duplet 
                 0.94 
               
               
                 PRE 
                 11294 
                 C 
                 5016 
                 PRE 
                 11294 
                 F 
                 5003 
                 Duplet 
                 0.94 
               
               
                 MOD 
                 10835 
                 C 
                 5013 
                 MOD 
                 10835 
                 G 
                 5015 
                 Duplet 
                 0.93 
               
               
                 PRE 
                 8115 
                 B 
                 5012 
                 PRE 
                 8115 
                 D 
                 5014 
                 Triplet 
                 0.93 
               
               
                 CON 
                 8416 
                 D 
                 5016 
                 CON 
                 8416 
                 B 
                 4997 
                 Duplet 
                 0.93 
               
               
                 PRE 
                 13159 
                 A 
                 5010 
                 PRE 
                 13159 
                 D 
                 5017 
                 Duplet 
                 0.93 
               
               
                 EAR 
                 11298 
                 D 
                 5003 
                 EAR 
                 11298 
                 H 
                 5013 
                 Triplet 
                 0.93 
               
               
                 MOD 
                 8192 
                 E 
                 5006 
                 MOD 
                 8192 
                 D 
                 4997 
                 Duplet 
                 0.93 
               
               
                 CON 
                 8421 
                 G 
                 5013 
                 CON 
                 8421 
                 G 
                 5014 
                 Triplet 
                 0.93 
               
               
                 PRE 
                 8115 
                 B 
                 5012 
                 PRE 
                 8115 
                 D 
                 5013 
                 Triplet 
                 0.93 
               
               
                 CON 
                 8421 
                 G 
                 5013 
                 CON 
                 8421 
                 G 
                 4998 
                 Triplet 
                 0.92 
               
               
                 PRE 
                 8115 
                 D 
                 5014 
                 PRE 
                 8115 
                 D 
                 5013 
                 Triplet 
                 0.91 
               
               
                 PRE 
                 13158 
                 D 
                 5011 
                 PRE 
                 13158 
                 H 
                 5002 
                 Duplet 
                 0.91 
               
               
                 PRE 
                 13266 
                 B 
                 5018 
                 PRE 
                 13266 
                 D 
                 5001 
                 Duplet 
                 0.91 
               
               
                 EAR 
                 11924 
                 B 
                 5004 
                 EAR 
                 11924 
                 F 
                 5015 
                 Duplet 
                 0.90 
               
               
                 CON 
                 8423 
                 A 
                 5004 
                 CON 
                 8423 
                 A 
                 5013 
                 Triplet 
                 0.89 
               
               
                 PRE 
                 12317 
                 D 
                 4998 
                 PRE 
                 12317 
                 E 
                 4997 
                 Duplet 
                 0.88 
               
               
                 CON 
                 8423 
                 A 
                 5004 
                 CON 
                 8423 
                 A 
                 5014 
                 Triplet 
                 0.88 
               
               
                 CON 
                 8423 
                 A 
                 5013 
                 CON 
                 8423 
                 A 
                 5014 
                 Triplet 
                 0.87 
               
               
                 ADV 
                 8361 
                 G 
                 5004 
                 ADV 
                 8361 
                 H 
                 5003 
                 Duplet 
                 0.87 
               
               
                 ADV 
                 13391 
                 B 
                 5003 
                 ADV 
                 13391 
                 B 
                 5010 
                 Duplet 
                 0.86 
               
               
                 CON 
                 8198 
                 G 
                 5001 
                   
                 Not-replicated 
                   
                   
                 Singlet 
                 0.84 
               
               
                 MOD 
                 8195 
                 A 
                 5014 
                   
                 Not-replicated 
                   
                   
                 Singlet 
                 0.84 
               
               
                 EAR 
                 10651 
                 B 
                 5007 
                 EAR 
                 10651 
                 C 
                 4997 
                 Duplet 
                 0.83 
               
               
                 PRE 
                 8227 
                 A 
                 5006 
                   
                 Not-replicated 
                   
                   
                 Singlet 
                 0.82 
               
               
                 CON 
                 13161 
                 C 
                 5010 
                 CON 
                 13161 
                 F 
                 5005 
                 Duplet 
                 0.81 
               
               
                 ADV 
                 8120 
                 E 
                 5008 
                 ADV 
                 8120 
                 A 
                 4997 
                 Duplet 
                 0.81 
               
               
                 MOD 
                 8386 
                 C 
                 4998 
                   
                 Not-replicated 
                   
                   
                 Singlet 
                 0.77 
               
               
                 CON 
                 10739 
                 A 
                 4998 
                   
                 Not-replicated 
                   
                   
                 Singlet 
                 0.77 
               
               
                 EAR 
                 8142 
                 G 
                 5005 
                   
                 Not-replicated 
                   
                   
                 Singlet 
                 0.77 
               
               
                 PRE 
                 8118 
                 F 
                 5006 
                   
                 Not-replicated 
                   
                   
                 Singlet 
                 0.76 
               
               
                 ADV 
                 13271 
                 A 
                 5000 
                 ADV 
                 13271 
                 H 
                 5000 
                 Duplet 
                 0.54 
               
               
                   
               
             
          
         
       
     
     The results of the correlation analysis of the Q10-Tris data set indicated that the majority of the replicate spectra were very similar. Indeed, 63 of the 80 comparisons resulted in values of r≧0.9. Of the 17 comparisons of replicate spectra that gave values of r&lt;0.9, seven were mean values of r for the non-replicated (singlet) spectra compared to the other spectra in the correlation matrix and these would perhaps be expected to be less than the direct comparisons of replicate spectra. Of the remaining 10 duplicate spectra that were correlated with r&lt;0.9, only one was particularly suspicious. The duplicates of sample 13271 were correlated with r=0.54. Closer inspection of this pair suggested that the spectrum acquired from position H of chip 5000 was visually dissimilar to the other spectra in the experiment and so this spectrum was excluded. The mean value of r across the correlation matrix for the remaining sample 13271 was 0.75, in line with mean values of the other non-replicated samples.  FIG. 6  shows scatter-plots of three replicate spectra in the Q10-Tris data set with correlation coefficients of 0.98 (1a), 0.90 (1b) and 0.54 (1c). In particular, the plots are:
     a) Duplicate spectra of sample 13342. The correlation coefficient of this pair is 0.98.   b) Duplicate spectra of sample 11924. The correlation coefficient of this pair is 0.90.   c) Duplicate spectra of sample 13271. The correlation coefficient of this pair is 0.54.   

     The correlations of replicate spectra in the CM10-AmAc and Q10-NaAc data sets involved only duplicate spectra and the Pearson correlation values are given in Tables 7 and 8, respectively. 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Pearson Correlation of replicate samples in the CM10-AmAc data set 
               
             
          
           
               
                 Group 
                 Sample 
                 Spot 
                 Chip 
                 Group 
                 Sample 
                 Spot 
                 Chip 
                 Correlation 
               
               
                   
               
             
          
           
               
                 ADV 
                 8113 
                 B 
                 1419 
                 ADV 
                 8113 
                 D 
                 1415 
                 0.98 
               
               
                 ADV 
                 8391 
                 D 
                 1616 
                 ADV 
                 8391 
                 D 
                 1412 
                 0.97 
               
               
                 ADV 
                 8120 
                 A 
                 1410 
                 ADV 
                 8120 
                 E 
                 1421 
                 0.97 
               
               
                 EAR 
                 11262 
                 B 
                 1615 
                 EAR 
                 11262 
                 B 
                 1617 
                 0.96 
               
               
                 CON 
                 10947 
                 C 
                 1416 
                 CON 
                 10947 
                 G 
                 1621 
                 0.96 
               
               
                 ADV 
                 8361 
                 G 
                 1417 
                 ADV 
                 8361 
                 H 
                 1416 
                 0.96 
               
               
                 MOD 
                 13165 
                 B 
                 1418 
                 MOD 
                 13165 
                 C 
                 1414 
                 0.96 
               
               
                 PRE 
                 13266 
                 B 
                 1622 
                 PRE 
                 13266 
                 D 
                 1414 
                 0.96 
               
               
                 PRE 
                 13262 
                 G 
                 1620 
                 PRE 
                 13262 
                 H 
                 1616 
                 0.96 
               
               
                 MOD 
                 10835 
                 C 
                 1617 
                 MOD 
                 10835 
                 G 
                 1619 
                 0.96 
               
               
                 MOD 
                 8119 
                 B 
                 1619 
                 MOD 
                 8119 
                 H 
                 1421 
                 0.96 
               
               
                 CON 
                 8416 
                 B 
                 1410 
                 CON 
                 8416 
                 D 
                 1620 
                 0.96 
               
               
                 ADV 
                 11841 
                 F 
                 1420 
                 ADV 
                 11841 
                 H 
                 1419 
                 0.96 
               
               
                 MOD 
                 8131 
                 E 
                 1614 
                 MOD 
                 8131 
                 E 
                 1416 
                 0.96 
               
               
                 MOD 
                 10945 
                 A 
                 1421 
                 MOD 
                 10945 
                 C 
                 1615 
                 0.95 
               
               
                 PRE 
                 8227 
                 A 
                 1419 
                 PRE 
                 8227 
                 G 
                 1618 
                 0.95 
               
               
                 EAR 
                 10837 
                 B 
                 1414 
                 EAR 
                 10837 
                 H 
                 1414 
                 0.95 
               
               
                 ADV 
                 8201 
                 H 
                 1614 
                 ADV 
                 8201 
                 H 
                 1410 
                 0.95 
               
               
                 PRE 
                 12581 
                 C 
                 1619 
                 PRE 
                 12581 
                 C 
                 1418 
                 0.95 
               
               
                 EAR 
                 13342 
                 C 
                 1413 
                 EAR 
                 13342 
                 G 
                 1421 
                 0.95 
               
               
                 EAR 
                 11924 
                 B 
                 1417 
                 EAR 
                 11924 
                 F 
                 1619 
                 0.94 
               
               
                 PRE 
                 12323 
                 D 
                 1417 
                 PRE 
                 12323 
                 F 
                 1622 
                 0.94 
               
               
                 MOD 
                 8126 
                 C 
                 1621 
                 MOD 
                 8126 
                 H 
                 1418 
                 0.94 
               
               
                 MOD 
                 8144 
                 B 
                 1620 
                 MOD 
                 8144 
                 G 
                 1616 
                 0.94 
               
               
                 PRE 
                 12360 
                 B 
                 1421 
                 PRE 
                 12360 
                 H 
                 1412 
                 0.94 
               
               
                 CON 
                 8421 
                 G 
                 1617 
                 CON 
                 8421 
                 G 
                 1411 
                 0.94 
               
               
                 PRE 
                 8118 
                 B 
                 1618 
                 PRE 
                 8118 
                 F 
                 1419 
                 0.94 
               
               
                 EAR 
                 8142 
                 E 
                 1618 
                 EAR 
                 8142 
                 G 
                 1418 
                 0.94 
               
               
                 MOD 
                 10866 
                 A 
                 1412 
                 MOD 
                 10866 
                 A 
                 1616 
                 0.93 
               
               
                 EAR 
                 11298 
                 D 
                 1416 
                 EAR 
                 11298 
                 H 
                 1617 
                 0.93 
               
               
                 CON 
                 10653 
                 A 
                 1414 
                 CON 
                 10653 
                 E 
                 1619 
                 0.93 
               
               
                 CON 
                 8413 
                 A 
                 1620 
                 CON 
                 8413 
                 E 
                 1415 
                 0.93 
               
               
                 PRE 
                 13159 
                 A 
                 1614 
                 PRE 
                 13159 
                 D 
                 1621 
                 0.93 
               
               
                 PRE 
                 11260 
                 B 
                 1412 
                 PRE 
                 11260 
                 B 
                 1415 
                 0.93 
               
               
                 EAR 
                 10651 
                 B 
                 1420 
                 EAR 
                 10651 
                 C 
                 1410 
                 0.93 
               
               
                 MOD 
                 8125 
                 A 
                 1622 
                 MOD 
                 8125 
                 F 
                 1620 
                 0.93 
               
               
                 MOD 
                 8192 
                 D 
                 1410 
                 MOD 
                 8192 
                 E 
                 1419 
                 0.93 
               
               
                 ADV 
                 13164 
                 F 
                 1615 
                 ADV 
                 13164 
                 F 
                 1414 
                 0.93 
               
               
                 CON 
                 10969 
                 A 
                 1621 
                 CON 
                 10969 
                 H 
                 1417 
                 0.93 
               
               
                 PRE 
                 12575 
                 E 
                 1413 
                 PRE 
                 12575 
                 F 
                 1614 
                 0.93 
               
               
                 MOD 
                 10868 
                 C 
                 1417 
                 MOD 
                 10868 
                 G 
                 1412 
                 0.92 
               
               
                 EAR 
                 8116 
                 B 
                 1621 
                 EAR 
                 8116 
                 F 
                 1616 
                 0.92 
               
               
                 CON 
                 11207 
                 G 
                 1615 
                 CON 
                 11207 
                 G 
                 1420 
                 0.92 
               
               
                 PRE 
                 12317 
                 D 
                 1411 
                 PRE 
                 12317 
                 E 
                 1410 
                 0.92 
               
               
                 PRE 
                 8115 
                 B 
                 1616 
                 PRE 
                 8115 
                 D 
                 1617 
                 0.92 
               
               
                 EAR 
                 12363 
                 D 
                 1622 
                 EAR 
                 12363 
                 F 
                 1415 
                 0.92 
               
               
                 MOD 
                 8195 
                 A 
                 1415 
                 MOD 
                 8195 
                 A 
                 1618 
                 0.92 
               
               
                 PRE 
                 12127 
                 D 
                 1420 
                 PRE 
                 12127 
                 H 
                 1619 
                 0.92 
               
               
                 ADV 
                 13271 
                 A 
                 1413 
                 ADV 
                 13271 
                 H 
                 1413 
                 0.91 
               
               
                 MOD 
                 10843 
                 C 
                 1420 
                 MOD 
                 10843 
                 D 
                 1413 
                 0.91 
               
               
                 EAR 
                 11289 
                 D 
                 1419 
                 EAR 
                 11289 
                 H 
                 1615 
                 0.91 
               
               
                 EAR 
                 11205 
                 D 
                 1614 
                 EAR 
                 11205 
                 H 
                 1621 
                 0.91 
               
               
                 EAR 
                 8117 
                 A 
                 1418 
                 EAR 
                 8117 
                 E 
                 1620 
                 0.91 
               
               
                 EAR 
                 12112 
                 A 
                 1619 
                 EAR 
                 12112 
                 F 
                 1412 
                 0.90 
               
               
                 CON 
                 10739 
                 A 
                 1411 
                 CON 
                 10739 
                 D 
                 1618 
                 0.90 
               
               
                 CON 
                 8114 
                 C 
                 1622 
                 CON 
                 8114 
                 E 
                 1412 
                 0.90 
               
               
                 PRE 
                 13158 
                 D 
                 1615 
                 PRE 
                 13158 
                 H 
                 1415 
                 0.89 
               
               
                 CON 
                 8198 
                 G 
                 1414 
                 CON 
                 8198 
                 H 
                 1618 
                 0.89 
               
               
                 ADV 
                 13272 
                 E 
                 1418 
                 ADV 
                 13272 
                 F 
                 1411 
                 0.89 
               
               
                 CON 
                 8423 
                 A 
                 1417 
                 CON 
                 8423 
                 A 
                 1617 
                 0.88 
               
               
                 MOD 
                 12492 
                 E 
                 1622 
                 MOD 
                 12492 
                 G 
                 1415 
                 0.88 
               
               
                 CON 
                 13166 
                 A 
                 1615 
                 CON 
                 13166 
                 E 
                 1616 
                 0.88 
               
               
                 MOD 
                 8386 
                 C 
                 1411 
                 MOD 
                 8386 
                 F 
                 1618 
                 0.88 
               
               
                 CON 
                 8358 
                 A 
                 1420 
                 CON 
                 8358 
                 C 
                 1419 
                 0.87 
               
               
                 CON 
                 13161 
                 C 
                 1614 
                 CON 
                 13161 
                 F 
                 1418 
                 0.87 
               
               
                 EAR 
                 8206 
                 A 
                 1416 
                 EAR 
                 8206 
                 H 
                 1411 
                 0.87 
               
               
                 EAR 
                 8355 
                 B 
                 1411 
                 EAR 
                 8355 
                 H 
                 1420 
                 0.87 
               
               
                 PRE 
                 11294 
                 C 
                 1620 
                 PRE 
                 11294 
                 F 
                 1416 
                 0.87 
               
               
                 CON 
                 10841 
                 B 
                 1413 
                 CON 
                 10841 
                 F 
                 1421 
                 0.85 
               
               
                 ADV 
                 13391 
                 B 
                 1416 
                 ADV 
                 13391 
                 B 
                 1614 
                 0.84 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Pearson Correlation of replicate samples in the Q10-NaAc data set 
               
             
          
           
               
                 Group 
                 Sample 
                 Spot 
                 Chip 
                 Group 
                 Sample 
                 Spot 
                 Chip 
                 Correlation 
               
               
                   
               
             
          
           
               
                 EAR 
                 11262 
                 B 
                 4974 
                 EAR 
                 11262 
                 B 
                 4976 
                 0.99 
               
               
                 PRE 
                 8115 
                 B 
                 4975 
                 PRE 
                 8115 
                 D 
                 4976 
                 0.98 
               
               
                 MOD 
                 8144 
                 B 
                 5360 
                 MOD 
                 8144 
                 G 
                 4975 
                 0.98 
               
               
                 EAR 
                 8116 
                 B 
                 4980 
                 EAR 
                 8116 
                 F 
                 4975 
                 0.98 
               
               
                 ADV 
                 8113 
                 B 
                 6514 
                 ADV 
                 8113 
                 D 
                 6510 
                 0.98 
               
               
                 PRE 
                 13159 
                 A 
                 4973 
                 PRE 
                 13159 
                 D 
                 4980 
                 0.98 
               
               
                 CON 
                 10841 
                 B 
                 6508 
                 CON 
                 10841 
                 F 
                 6516 
                 0.98 
               
               
                 MOD 
                 8192 
                 D 
                 6505 
                 MOD 
                 8192 
                 E 
                 6514 
                 0.98 
               
               
                 CON 
                 10739 
                 A 
                 6506 
                 CON 
                 10739 
                 D 
                 4977 
                 0.97 
               
               
                 EAR 
                 12363 
                 D 
                 4981 
                 EAR 
                 12363 
                 F 
                 6510 
                 0.97 
               
               
                 MOD 
                 10835 
                 C 
                 4976 
                 MOD 
                 10835 
                 G 
                 4978 
                 0.97 
               
               
                 MOD 
                 10866 
                 A 
                 6507 
                 MOD 
                 10866 
                 A 
                 4975 
                 0.97 
               
               
                 ADV 
                 11841 
                 F 
                 6515 
                 ADV 
                 11841 
                 H 
                 6514 
                 0.97 
               
               
                 ADV 
                 8361 
                 G 
                 6502 
                 ADV 
                 8361 
                 H 
                 6511 
                 0.97 
               
               
                 MOD 
                 8126 
                 C 
                 4980 
                 MOD 
                 8126 
                 H 
                 6513 
                 0.97 
               
               
                 CON 
                 8198 
                 G 
                 6509 
                 CON 
                 8198 
                 H 
                 4977 
                 0.97 
               
               
                 PRE 
                 11294 
                 C 
                 5360 
                 PRE 
                 11294 
                 F 
                 6511 
                 0.97 
               
               
                 EAR 
                 10651 
                 B 
                 6515 
                 EAR 
                 10651 
                 C 
                 6505 
                 0.97 
               
               
                 CON 
                 8416 
                 B 
                 6505 
                 CON 
                 8416 
                 D 
                 5360 
                 0.97 
               
               
                 PRE 
                 13158 
                 D 
                 4974 
                 PRE 
                 13158 
                 H 
                 6510 
                 0.97 
               
               
                 MOD 
                 13165 
                 B 
                 6513 
                 MOD 
                 13165 
                 C 
                 6509 
                 0.96 
               
               
                 MOD 
                 8386 
                 C 
                 6506 
                 MOD 
                 8386 
                 F 
                 4977 
                 0.96 
               
               
                 CON 
                 11207 
                 G 
                 4974 
                 CON 
                 11207 
                 G 
                 6515 
                 0.96 
               
               
                 EAR 
                 12112 
                 A 
                 4978 
                 EAR 
                 12112 
                 F 
                 6507 
                 0.96 
               
               
                 PRE 
                 8227 
                 A 
                 6514 
                 PRE 
                 8227 
                 G 
                 4977 
                 0.96 
               
               
                 MOD 
                 10843 
                 C 
                 6515 
                 MOD 
                 10843 
                 D 
                 6508 
                 0.96 
               
               
                 ADV 
                 13271 
                 A 
                 6508 
                 ADV 
                 13271 
                 H 
                 6508 
                 0.96 
               
               
                 CON 
                 8114 
                 C 
                 4981 
                 CON 
                 8114 
                 E 
                 6507 
                 0.96 
               
               
                 CON 
                 8358 
                 A 
                 6515 
                 CON 
                 8358 
                 C 
                 6514 
                 0.96 
               
               
                 CON 
                 10969 
                 A 
                 4980 
                 CON 
                 10969 
                 H 
                 6502 
                 0.96 
               
               
                 MOD 
                 8125 
                 A 
                 4981 
                 MOD 
                 8125 
                 F 
                 5360 
                 0.96 
               
               
                 EAR 
                 8117 
                 A 
                 6513 
                 EAR 
                 8117 
                 E 
                 5360 
                 0.96 
               
               
                 MOD 
                 8131 
                 E 
                 4973 
                 MOD 
                 8131 
                 E 
                 6511 
                 0.96 
               
               
                 ADV 
                 13164 
                 F 
                 4974 
                 ADV 
                 13164 
                 F 
                 6509 
                 0.95 
               
               
                 PRE 
                 12575 
                 E 
                 6508 
                 PRE 
                 12575 
                 F 
                 4973 
                 0.95 
               
               
                 CON 
                 8421 
                 G 
                 4976 
                 CON 
                 8421 
                 G 
                 6506 
                 0.95 
               
               
                 PRE 
                 12317 
                 D 
                 6506 
                 PRE 
                 12317 
                 E 
                 6505 
                 0.95 
               
               
                 ADV 
                 8201 
                 H 
                 4973 
                 ADV 
                 8201 
                 H 
                 6505 
                 0.95 
               
               
                 CON 
                 8423 
                 A 
                 6502 
                 CON 
                 8423 
                 A 
                 4976 
                 0.95 
               
               
                 PRE 
                 12360 
                 B 
                 6516 
                 PRE 
                 12360 
                 H 
                 6507 
                 0.95 
               
               
                 PRE 
                 13262 
                 G 
                 5360 
                 PRE 
                 13262 
                 H 
                 4975 
                 0.94 
               
               
                 ADV 
                 8120 
                 A 
                 6505 
                 ADV 
                 8120 
                 E 
                 6516 
                 0.94 
               
               
                 CON 
                 13166 
                 A 
                 4974 
                 CON 
                 13166 
                 E 
                 4975 
                 0.94 
               
               
                 EAR 
                 11298 
                 D 
                 6511 
                 EAR 
                 11298 
                 H 
                 4976 
                 0.94 
               
               
                 EAR 
                 8206 
                 A 
                 6511 
                 EAR 
                 8206 
                 H 
                 6506 
                 0.94 
               
               
                 EAR 
                 11924 
                 B 
                 6502 
                 EAR 
                 11924 
                 F 
                 4978 
                 0.94 
               
               
                 CON 
                 8413 
                 A 
                 5360 
                 CON 
                 8413 
                 E 
                 6510 
                 0.94 
               
               
                 EAR 
                 11205 
                 D 
                 4973 
                 EAR 
                 11205 
                 H 
                 4980 
                 0.94 
               
               
                 PRE 
                 11260 
                 B 
                 6507 
                 PRE 
                 11260 
                 B 
                 6510 
                 0.93 
               
               
                 MOD 
                 10945 
                 A 
                 6516 
                 MOD 
                 10945 
                 C 
                 4974 
                 0.93 
               
               
                 EAR 
                 10837 
                 B 
                 6509 
                 EAR 
                 10837 
                 H 
                 6509 
                 0.93 
               
               
                 MOD 
                 12492 
                 E 
                 4981 
                 MOD 
                 12492 
                 G 
                 6510 
                 0.93 
               
               
                 MOD 
                 8119 
                 B 
                 4978 
                 MOD 
                 8119 
                 H 
                 6516 
                 0.93 
               
               
                 PRE 
                 12127 
                 D 
                 6515 
                 PRE 
                 12127 
                 H 
                 4978 
                 0.93 
               
               
                 CON 
                 10653 
                 A 
                 6509 
                 CON 
                 10653 
                 E 
                 4978 
                 0.93 
               
               
                 EAR 
                 11289 
                 D 
                 6514 
                 EAR 
                 11289 
                 H 
                 4974 
                 0.92 
               
               
                 MOD 
                 8195 
                 A 
                 6510 
                 MOD 
                 8195 
                 A 
                 4977 
                 0.92 
               
               
                 ADV 
                 13272 
                 E 
                 6513 
                 ADV 
                 13272 
                 F 
                 6506 
                 0.92 
               
               
                 CON 
                 13161 
                 C 
                 4973 
                 CON 
                 13161 
                 F 
                 6513 
                 0.91 
               
               
                 EAR 
                 8355 
                 B 
                 6506 
                 EAR 
                 8355 
                 H 
                 6515 
                 0.91 
               
               
                 MOD 
                 10868 
                 C 
                 6502 
                 MOD 
                 10868 
                 G 
                 6507 
                 0.91 
               
               
                 PRE 
                 13266 
                 B 
                 4981 
                 PRE 
                 13266 
                 D 
                 6509 
                 0.90 
               
               
                 PRE 
                 12581 
                 C 
                 4978 
                 PRE 
                 12581 
                 C 
                 6513 
                 0.90 
               
               
                 ADV 
                 13391 
                 B 
                 6511 
                 ADV 
                 13391 
                 B 
                 4973 
                 0.89 
               
               
                 PRE 
                 12323 
                 D 
                 6502 
                 PRE 
                 12323 
                 F 
                 4981 
                 0.88 
               
               
                 ADV 
                 8391 
                 D 
                 4975 
                 ADV 
                 8391 
                 D 
                 6507 
                 0.87 
               
               
                 EAR 
                 13342 
                 C 
                 6508 
                 EAR 
                 13342 
                 G 
                 6516 
                 0.87 
               
               
                 EAR 
                 8142 
                 E 
                 4977 
                 EAR 
                 8142 
                 G 
                 6513 
                 0.85 
               
               
                 PRE 
                 8118 
                 B 
                 4977 
                 PRE 
                 8118 
                 F 
                 6514 
                 0.83 
               
               
                   
               
             
          
         
       
     
     In the CM10-AmAc data set, the values of Pearson correlation values for the duplicate spectra ranged from 0.98 to 0.84, with 56 of the 70 duplicates being correlated with r≧0.90. In the Q10-NaAc data set, the Pearson correlation values ranged from 0.99 to 0.83, with 63 of the 69 duplicates being correlated with r≧0.90. No spectra were excluded from these data sets on the basis of the correlation analysis. 
     Averaging: To improve the reliability of the measurements of peaks in the SELDI profiles, averages (means) were calculated from the available replicates. This has previously been shown in our laboratory to improve correlations between a set of spectra comprising biological replicates when averages of pairs are taken to represent the sample. For the data analysis, averaged data were used in place of the original replicates. This is particularly important because it avoids giving an over-estimate of the degrees of freedom in the statistical hypothesis tests, as would occur when replicate samples are used as if they were independent biological samples. 
     Statistical Hypothesis Testing 
     Several related methods were used for univariate data analysis of the quantile normalised and averaged data set. These can broadly be divided into tests for the assumption that all the means are equal, and multiple comparisons procedures that test the equality of the means of individual pairs of groups. Additionally, a test for homogeneity of variances was performed before testing the means to determine the appropriate set of tests to perform. 
     In order to test the important assumption of ANOVA that the groups have equal variance, Levene&#39;s test was used at the 95% level. If Levene&#39;s test returned a p-value of &gt;0.05, the alternative hypothesis was rejected and the groups were assumed to have equal variance. When equal variance was assumed, one-way ANOVA was used to test the equality of group means. When equal variance could not be assumed (i.e. when Levene&#39;s test returned a p-value of &lt;0.05) Welch&#39;s test for equality of means was used as a more robust alternative. Both the one-way ANOVA and Welch&#39;s test were preformed at the 95% level. 
     When the group means were found to be unequal, one of two tests were used to test all pairs of groups in the data sets. If the means were found to be unequal using the one-way ANOVA test, Tukey&#39;s honestly significant difference (HSD) was used to compare all groups. If the means were found to be unequal using Welch&#39;s test, then Tamhane&#39;s T2 was employed to compare all groups. Both multiple comparisons methods were performed at the 95% level. 
     Table 9 shows information relating to the peaks found to have statistically significant differences in the means of the five groups (CON, PRE, EAR, MOD and ADV). 
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Peaks found to be statistically different in the Q10-Tris, Q10-NaAc or CM10- 
               
               
                 AmAc data sets using the univariate tests. 
               
             
          
           
               
                 Master 
                   
                   
                   
                 Equality 
                   
               
               
                 Peak No. 
                 Data set 
                 Peak m/z 
                 Peak m/z 95% CI 
                 of means 
                 Group differences 
               
               
                   
               
             
          
           
               
                 1 
                 Q10-Tris 
                 3564.76 
                 3555.50-3574.22 
                 0.018 a   
                 CON ≠ ADV (1.25-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.27-fold decreased in ADV) 
               
               
                 2 
                 CM10-AmAc 
                 3662.78 
                 3656.58-3679.43 
                 0.002 a   
                 CON ≠ ADV (1.57-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.37-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (1.51-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (1.48-fold decreased in ADV) 
               
               
                 3 
                 CM10-AmAc 
                 4227.05 
                 4206.08-4228.74 
                 0.040 a   
                 PRE ≠ ADV (1.74-fold decreased in ADV) 
               
               
                 4 
                 Q10-NaAc 
                 4296.42 
                 4287.60-4301.56 
                 0.011 a   
                 PRE ≠ ADV (3.85-fold decreased in ADV) 
               
               
                 5 
                 Q10-NaAc 
                 4357.75 
                 4351.19-4364.63 
                 0.023 a   
                 CON ≠ ADV (1.90-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.95-fold decreased in ADV) 
               
               
                 6 
                 Q10-Tris 
                 4371.83 
                 4360.58-4376.90 
                 0.047 a   
                 CON ≠ ADV (1.38-fold increased in ADV) 
               
               
                 7 
                 Q10-Tris 
                 4479.08 
                 4471.89-4481.98 
                 0.013 b   
                 CON ≠ ADV(1.78-fold increased in ADV) 
               
               
                 8 
                 Q10-NaAc 
                 4720.37 
                 4716.23-4724.48 
                 0.000 a   
                 CON ≠ ADV (2.09-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.91-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (1.87-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (2.06-fold decreased in ADV) 
               
               
                 8 
                 Q10-Tris 
                 4721.02 
                 4710.43-4726.35 
                 0.007 b   
                 CON ≠ ADV (1.33-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD ≠ ADV (1.52-fold decreased in ADV) 
               
               
                 9 
                 Q10-NaAc 
                 5760.90 
                 5751.18-5778.87 
                 0.035 a   
                 EAR ≠ ADV (1.57-fold decreased in ADV) 
               
               
                 10 
                 CM10-AmAc 
                 5966.63 
                 5960.49-5981.23 
                 0.005 a   
                 CON ≠ EAR (1.31-fold increased in EAR) 
               
               
                   
                   
                   
                   
                   
                 MOD ≠ EAR (1.35-fold increased in EAR) 
               
               
                 11 
                 Q10-NaAc 
                 6523.63 
                 6515.57-6540.55 
                 0.046 b   
                 PRE ≠ ADV (3.29-fold decreased in ADV) 
               
               
                 12 
                 CM10-AmAc 
                 6919.51 
                 6913.11-6927.14 
                 0.018 a   
                 CON ≠ EAR (1.16-fold decreased in EAR) 
               
               
                 13 
                 Q10-NaAc 
                 6985.41 
                 6983.66-7011.20 
                 0.008 a   
                 CON ≠ ADV (2.54-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (2.62-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (2.32-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (2.37-fold decreased in ADV) 
               
               
                 14 
                 CM10-AmAc 
                 7034.90 
                 7030.01-7043.33 
                 0.008 a   
                 CON ≠ ADV (1.48-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.54-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (1.47-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (1.42-fold decreased in ADV) 
               
               
                 15 
                 CM10-AmAc 
                 7080.59 
                 7067.18-7087.69 
                 0.007 a   
                 CON ≠ ADV (1.39-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.38-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (1.38-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (2.08-fold decreased in ADV) 
               
               
                 16 
                 Q10-NaAc 
                 7624.59 
                 7605.45-7637.52 
                 0.005 b   
                 MOD ≠ ADV (1.48-fold decreased in ADV) 
               
               
                 17 
                 CM10-AmAc 
                 8139.10 
                 8133.70-8149.02 
                 0.005 a   
                 CON ≠ ADV (1.85-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (2.17-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (1.88-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (2.08-fold increased in ADV) 
               
               
                 18 
                 CM10-AmAc 
                 8208.41 
                 8204.81-8224.70 
                 0.016 a   
                 CON ≠ ADV (1.87-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.92-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (1.97-fold increased in ADV) 
               
               
                 19 
                 CM10-AmAc 
                 8251.49 
                 8228.44-8251.50 
                 0.001 b   
                 CON ≠ ADV (2.00-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (2.07-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (1.72-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (2.44-fold increased in ADV) 
               
               
                 20 
                 Q10-NaAc 
                 8466.00 
                 8456.99-8472.79 
                 0.013 b   
                 CON ≠ ADV (4.32-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 (p &lt; 0.15) c   
               
               
                 21 
                 Q10-NaAc 
                 8763.16 
                 8760.65-8775.33 
                 0.030 b   
                 CON ≠ ADV (1.59-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.71-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (1.70-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (1.67-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 (p &lt; 0.20) c   
               
               
                 22 
                 Q10-NaAc 
                 9135.76 
                 9124.91-9140.55 
                 0.028 b   
                 MOD ≠ ADV (3.59-fold decreased in ADV) 
               
               
                 23 
                 Q10-NaAc 
                 9632.25 
                 9624.52-9652.90 
                 0.027 b   
                 CON ≠ ADV (2.81-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD ≠ ADV (2.77-fold decreased in ADV) 
               
               
                 24 
                 Q10-NaAc 
                 9936.86 
                 9912.33-9946.84 
                 0.035 b   
                 EAR ≠ ADV (1.94-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (1.80-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 (p &lt; 0.19) c   
               
               
                 25 
                 Q10-NaAc 
                 10450.60 
                 10425.49-10476.21 
                 0.039 b   
                 EAR ≠ ADV (1.85-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (1.61-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 (p &lt; 0.13) c   
               
               
                 26 
                 Q10-Tris 
                 11533.31 
                 11496.71-11559.00 
                 0.020 a   
                 PRE ≠ ADV (2.59-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD ≠ ADV (2.32-fold increased in ADV) 
               
               
                 27 
                 CM10-AmAc 
                 15964.13 
                 15943.28-15988.41 
                 0.004 a   
                 CON ≠ ADV (1.94-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (2.22-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (2.03-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (2.07-fold increased in ADV) 
               
               
                 28 
                 CM10-AmAc 
                 16117.87 
                 16094.26-16140.52 
                 0.011 a   
                 CON ≠ ADV (1.90-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (2.07-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (2.11-fold increased in ADV) 
               
               
                 29 
                 CM10-AmAc 
                 16320.30 
                 16296.87-16349.18 
                 0.003 b   
                 CON ≠ ADV (2.36-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (2.73-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (1.97-fold increased in ADV) 
               
               
                   
                   
                   
                   
                   
                 MOD≠ ADV (2.99-fold increased in ADV) 
               
               
                 30 
                 Q10-NaAc 
                 21018.23 
                 20980.22-21073.08 
                 0.020 b   
                 CON ≠ ADV (1.30-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.20-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 (p &lt; 0.14) c   
               
               
                 31 
                 Q10-Tris 
                 37324.17 
                 37166.16-37590.06 
                 0.000 b   
                 CON ≠ ADV (1.33-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.56-fold decreased in ADV) 
               
               
                 31 
                 Q10-NaAc 
                 37415.98 
                 36906.53-37633.64 
                 0.037 b   
                 CON ≠ ADV (1.29-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 PRE ≠ ADV (1.39-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 EAR ≠ ADV (1.15-fold decreased in ADV) 
               
               
                   
                   
                   
                   
                   
                 (p &lt; 0.17) c   
               
               
                 32 
                 Q10-Tris 
                 41829.80 
                 41611.37-42059.30 
                 0.004 a   
                 PRE ≠ ADV (1.53-fold decreased in ADV) 
               
               
                 33 
                 Q10-NaAc 
                 50472.78 
                 50056.68-50933.57 
                 0.018 b   
                 CON ≠ EAR (1.42-fold increased in EAR) 
               
               
                 34 
                 Q10-Tris 
                 56159.58 
                 55976.01-56218.65 
                 0.008 a   
                 PRE ≠ ADV (1.54-fold decreased in ADV) 
               
               
                   
               
               
                   a Group means were found to be unequal by one-way ANOVA. 
               
               
                   b Group means were found to be unequal by Welch&#39;s test. 
               
               
                   c Group means were unequal by Welch&#39;s test but no individual groups were different at the 95% level by Tamhane&#39;s test. 
               
             
          
         
       
     
     The groups significant at the 80% level for Tamhane&#39;s test are reported. 
     In total, there were 32 peaks found to have statistically significant differences in the means of all groups in the three data sets. In the Q10-Tris data set, there were eight peaks showing statistically significant differences in the mean peak intensity of the groups as a whole. In the Q10-NaAc data set, there were 16 peaks displaying statistically significant differences in the mean peak intensity of the groups. In the CM10-AmAc data set, there were 12 peaks showing statistically significant differences in the mean peak intensity of the groups. Of these peaks differing between the groups, there was some overlap between the three data sets. Namely, peaks 8 and 31 both showed a statistically significant difference between the mean peak intensity of the groups in both the Q10-Tris and Q10-NaAc data sets. Some group comparisons in the Q10-NaAc data set found using Welch&#39;s test did not show any significant differences using Tamhane&#39;s T2 at the 95% level, presumably because of the conservative nature of this multiple comparison test. Where this was the case, groups differing at the 80% level were given as the groups most likely to cause the difference detected by Welch&#39;s test. 
     For each statistically significant group difference, a fold-change between the means of the groups was calculated and displayed in Table 9. There were a total of 59 individual group differences with mean peak intensity fold-changes of greater that 1.5 and these derived from 29 distinct peaks. These changes therefore likely represent the most robust and important differences between the groups. 
     A prominent feature of the group differences listed in Table 9 is that the ADV group is the most often statistically different group compared to the other groups. There were a total of 82 individual group differences found and of these, 78 were a comparison of the ADV group with one of the other groups. This result does not necessarily imply that the changes observed only occurred in the advanced stages of HD, only that if the changes did progress with the disease that they were not large enough to be of statistical significance by the tests used.  FIG. 8  shows box and whisker plots summarising the distributions of the peak intensities of the statistically differing peaks in each group. For each peak, the data set and m/z value are given along with a box and whisker plot showing the distribution of values within each group. The groups are labelled  1  (CON),  2  (PRE),  3  (EAR),  4  (MOD) and  5  (ADV). 
     SUMMARY 
     The SELDI analysis of samples from the CON and HD groups detected in excess of 200 peaks in across three data sets. Of these peaks, 36 were found to be statistically different between one or more groups and two of these peaks were found to differ in both the Q10-Tris and Q10-NaAc data sets, giving 34 individually changing peaks. The number and overlap of the statistically different peaks in the three experimental data sets is displayed graphically in the form of a Venn diagram in  FIG. 7 . Of these 34 distinct peaks, 29 showed fold-changes between one or more groups of greater than 1.5-fold. 
     Further results are shown below in Table 10. This is a summary of all the proteins we have identified in material extracted from the SELDI chips. Any of the peaks we have observed in the SELDI profiles originate from any of the proteins listed in the table, either as the expected mature proteins or fragments of the proteins. This list of proteins and any fragments thereof thus constitute sequences that would feasibly generate the m/z values we see in the SELDI spectra. 
     
       
         
               
               
               
             
           
               
                   
                 TABLE 10 
               
               
                   
                   
               
               
                   
                 Swiss Prot 
                   
               
               
                   
                 accession number 
                 Protein name 
               
               
                   
                   
               
             
             
               
                   
                 P00738 
                 Haptoglobin precursor 
               
               
                   
                 P01009 
                 Alpha-1-antitrypsin precursor 
               
               
                   
                 P01024 
                 Complement C3 precursor 
               
               
                   
                 P01620 
                 Ig kappa chain V-III region 
               
               
                   
                 P01834 
                 Ig kappa chain C region 
               
               
                   
                 P01842 
                 Ig lambda chain C regions 
               
               
                   
                 P01857 
                 Ig gamma-1 chain C region 
               
               
                   
                 P01859 
                 Ig gamma-2 chain C region 
               
               
                   
                 P01876 
                 Ig alpha-1 chain C region 
               
               
                   
                 P02647 
                 Apolipoprotein A-I precursor 
               
               
                   
                 P02649 
                 Apolipoprotein E precursor 
               
               
                   
                 P02652 
                 Apolipoprotein A-II precursor 
               
               
                   
                 P02655 
                 Apolipoprotein C-II precursor 
               
               
                   
                 P02656 
                 Apolipoprotein C-III precursor 
               
               
                   
                 P02671 
                 Fibrinogen alpha/alpha-E chain precursor 
               
               
                   
                 P02763 
                 Alpha-1-acid glycoprotein 1 precursor 
               
               
                   
                 P02766 
                 Transthyretin precursor 
               
               
                   
                 P02768 
                 Serum albumin precursor 
               
               
                   
                 P02787 
                 Serotransferrin precursor 
               
               
                   
                 P04196 
                 Histidine-rich glycoprotein precursor 
               
               
                   
                 P06727 
                 Apolipoprotein A-IV precursor 
               
               
                   
                 P19652 
                 Alpha-1-acid glycoprotein 2 precursor 
               
               
                   
                 P68871/P02042 
                 Hemoglobin beta chain/Hemoglobin delta chain 
               
               
                   
                 P10909 
                 Clusterin 
               
               
                   
                   
               
             
          
         
       
     
     We have correlated 6 of the 34 peak m/z observed in SELDI to the sequences indicated below. The following Table 11 refers to Master peak numbers indicated in Table 9 and correlates SELDI peak m/z with protein sequence information from LC/MS/MS results. 
     
       
         
               
               
               
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                   
                   
                   
                   
                 Amino acid 
               
               
                 Master 
                   
                   
                 Swiss Prot 
                 Residues (as 
               
               
                 Peak 
                 Peak 
                   
                 Accession 
                 given in Swiss Prot 
               
               
                 No. 
                 m/z 
                 Protein 
                 No. 
                 database entry 
               
               
                   
               
             
             
               
                 13 
                 6985.41 
                 Apolipoprotein A-II 
                 P02652 
                 39-100 
               
               
                 16 
                 7624.59 
                 Apolipoprotein A-II 
                 P02652 
                 34-100 
               
               
                 18 
                 8208.41 
                 Apolipoprotein C-II 
                 P02655 
                 29-101 
               
               
                 19 
                 8251.49 
                 Apolipoprotein A-II 
                 P02652 
                 28-100 
               
               
                 20 
                 8466.00 
                 Apolipoprotein C-II 
                 P02655 
                 27-101 
               
               
                 21 
                 8763.16 
                 Apolipoprotein C-III 
                 P02656 
                 21-99  
               
               
                   
                   
                   
                   
                 (Expected Mature 
               
               
                   
                   
                   
                   
                 form) 
               
               
                   
               
             
          
         
       
     
     Each of the above-cited publications and database references is herein incorporated by reference to the extent to which it is relied on herein.