bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and classification of autoantibody repertoires (Western blots) with a pattern recognition algorithm by an artificial neural network
Authors: Grus Franz‐Hermann , Zimmermann Christoph W.
Published: May 2005

The screening of sera for autoantibodies with Western blots reveals complex repertoires. The composition of such repertoires depends on genetic control of autoantibody‐producing cells, the individual's history of exposure to its own and to foreign antigens, and also on the presence of autoimmune diseases. Our method shows how staining patterns of Western blots can be recoded as binary or grey‐value vectors. Vectors are transferred to artificial neural networks for learning. Artificial neural networks are able to recognize group‐specific antibody binding patterns. Staining patterns can be attributed to diagnostic groups. This may support diagnostic procedures.
The effect of column length, applied voltage, gel type, and concentration on the capillary electrophoresis separation of DNA fragments and polymerase chain reaction products
Authors: Issaq Haleem J. , Chan King C. , Muschik Gary M.
Published: May 2005

This work examines the effect of different parameters on migration time, resolution, and speed of analysis of DNA fragments and PCR products. These parameters include column length, applied voltage, gel type and concentration, and buffer ionic strength. Our results indicate that 1 cm capillary at an applied voltage of 185 V/cm, filled with commercial gel, was adequate for the separation of small DNA fragments in under 1 min. Resolution of large fragments is directly proportional to column length at the same field strength. Also, resolution of large fragments is higher (better) at lower field strength at constant column length. Analysis is fastest (high throughput) using a short capillary and moderate field strength (200 v/cm). CE using a single short capillary (2–7 cm) is comparable to slab gel in throughput, but more economical. The Sigma DNA buffer and hydroxyethyl cellulose liquid gel gave equivalent results in terms of resolution and reproducibility. The Sigma DNA replaceable gel gave reproducible results when used as received or diluted at 60%. In our hands hydroxyethyl cellulose gave more reproducible results than polyacrylamide gel.
Proteome research: Complementarity and limitations with respect to the RNA and DNA worlds
Authors: Humphery‐Smith Ian , Cordwell Stuart J. , Blackstock Walter P.
Published: May 2005

A methodological overview of proteome analysis is provided along with details of efforts to achieve high‐throughput screening (HTS) of protein samples derived from two‐dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700–1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)‐based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time‐of‐flight (MALDI‐TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI‐TOF‐mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene‐product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half‐life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene‐products on biochemical flux; the influence of the environment, cell‐cycle, stress and disease on gene‐products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene‐products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large‐scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two‐dimensional (2‐D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.
Additional approaches to DNA typing of skeletal remains: The search for “missing” persons killed during the last dictatorship in Argentina
Authors: Corach Daniel , Sala Andrea , Penacino Gustavo , Iannucci Nancy , Bernardi Patricia , Doretti Mercedes , Fondebrider Luis , Ginarte Anahi , Inchaurregui Alejandro , Somigliana Carlos , Turner Silvana , Hagelberg Erika
Published: May 2005

DNA typing techniques are among the most advanced tools for human identification and can contribute to the identification of poorly preserved skeletal remains. Ten thousand people are thought to have been killed during the last dictatorship in Argentina (1976–1983) and there are few official records on the identity of the victims or the location of burials. A mass grave containing 340 skeletons was excavated using archeological methods. A small number of individuals was identified by traditional forensic methods and one family group by mitochondrial DNA (mtDNA) analysis. Due to the lack of antemortem physical information on many of the victims, the application of molecular methods is imperative to speed up the identification process. We have tested two molecular screening methods, Y chromosome‐specific short tandem repeats (DYS19, DYS385, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS393) and amplification of autosomal microsatellites using nested primers. These methods can complement solely matrilineal mtDNA sequence data in the identification of “missing” persons.
Molecular advances in understanding social insect population structure
Authors: Crozier Ross H. , Oldroyd Ben P. , Tay W. Tek , Kaufmann Bernard E. , Johnson Rebecca N. , Carew Melissa E. , Jennings Kim M.
Published: May 2005

Social insects present many phenomena seen in all organisms but in more extreme forms and with larger sample sizes than those observable in most natural populations of vertebrates. Microsatellites are proving very much more informative than allozymes for the analysis of population biological problems, and prolifically polymorphic markers are fairly readily developed. In addition, the male‐haploid genetic system of many social insects facilitates genetic analysis. The ability to amplify DNA from sperm stored in a female's sperm storage device enables the determination of mating types long after the death of the short‐lived males, in addition to information on the degree of mixing of sperm from different males. Mitochondrial (mt) DNA sequences are also proving important, not only in phylogenetic studies but also in molecular population genetics, as a tracer of female movements. Mitochondrial markers have definitively shown the movement of females between colonies, challenging models giving exclusive primacy to kin selection as the explanation for multi‐queen colonies, in Australian meat ants,Iridomyrmex purpureus, and the arid‐zone queenless antRhytidoponerasp. 12. Microsatellite and mtDNA variation are being studied inCamponotus consobrinussugar ants, showing an unexpected diversity of complexity in colony structure, and microsatellites have shown that transfer of ants between nests of the weaver antPolyrhachis doddimust be slight, despite an apparent lack of hostility.
Analysis of carbohydrate deficient transferrin by capillary zone electrophoresis
Authors: Prasad Rajani , Stout Robert L. , Coffin David , Smith James
Published: May 2005

We report a capillary zone electrophoresis method to separate the various sialylated isoforms of transferrin. The separation is carried out under nondenaturing conditions and at basic pH. Under these conditions, transferrin exhibits two major and three minor peaks. Plasma samples from a population consuming varying amounts of alcohol at different intervals were studied. A cut‐off value of 3% carbohydrate deficient transferrin (CDT: disialo, monosialo, and asialo transferrin), results in a clinical sensitivity of 88% in a population consuming at least 70 g/day alcohol for a minimum of two weeks. The sensitivity dropped significantly in a population consuming less than 70 g/day. This confirms previous reports of CDT as a specific marker for significant and chronic use of alcohol. Capillary electrophoresis offers an alternative method with respect to analysis time and throughput in the clinical laboratory.
Simultaneous determination of S‐adenosylmethionine and S‐adenosylhomocysteine by capillary zone electrophoresis
Authors: Pañak Karina C. , Giorgieri Sergio A. , Díaz Luis E. , Ruiz Oscar A.
Published: May 2005

A reproducible, rapid procedure for the simultaneous quantitative separation of S‐adenosylmethionine and S‐adenosylhomocysteine by capillary zone electrophoresis has been developed. Separations were performed by using an uncoated capillary of 60 cm effective length and 50 μm ID, 40 mM sodium phosphate buffer, pH 2.50, as background electrolyte solution, and 30 kV. On‐line detection was carried out at 254 nm. Under the conditions selected we resolved a standard solution containing S‐adenosylmethionine and S‐adenosylhomocysteine in a run time shorter than 8 min. A mass detection limit in the range of 10 fmol was achieved. Adenosyl‐L‐methionine, S[methyl‐3H] has also been assayed under the same experimental conditions. Other related compounds did not show interference, including those derived from the hydrolysis of S‐adenosylmethionine. The present method allows simultaneous determination of these compounds, which play an important role in many microbiological and enzymatic research studies.
Column technology in capillary electrophoresis and capillary electrochromatography
Authors: Pesek Joseph J. , Matyska Maria T.
Published: May 2005

A review of column technology in capillary electrophoresis (CE) including wall modification processes and open tubular as well as packed column formats in capillary electrochromatography (CEC) are presented. There are many approaches which can be used to solve separation problems which provide higher efficiency and/or shorter analysis times in comparison to other chromatographic techniques. However, both CE and CEC are still relatively undeveloped in comparison to a more mature method such as high performance liquid chromatography (HPLC) and improvement in column technology is changing rapidly.
Polymeric separation media for capillary electrophoresis of nucleic acids
Authors: Sunada Wade M. , Blanch Harvey W.
Published: May 2005

The choice of polymer matrix for separating nucleic acids by capillary electro‐phoresis has often been arbitrary. However, considerable research in the area has led to a wealth of data exploring the key parameters of the polymer matrix that affect nucleic acid separations: polymer type, polymer molecular mass, polymer concentration, temperature, and buffer components and additives. Using this information, it is possible to use rational methods of choosing a good polymer matrix for a particular application. Further research into the properties of the mechanism of separation in polymer solutions, as well as the polymer matrix and other solution components will lead to even more efficient separations.
Chemiluminescence detection in capillary electrophoresis
Authors: Staller Tracy D. , Sepaniak Michael J.
Published: May 2005

Capillary electrophoresis (CE) and related techniques yield highly efficient separations while requiring only minute amounts of sample. Thus, these techniques are amenable to analyses of complex samples in diverse matrices and in situations where sample is extremely limited. The constraints of on‐column detection generally result in poor detection limits and have reduced the overall application of CE. One logical approach to increased sensitivity in CE detection has been the development of chemiluminescence (CL)‐based detectors. The current state of post‐column detector development, CL applications, and limitations of the technique are reported herein.
Capillary electrophoretic enantioseparations using macrocyclic antibiotics as chiral selectors
Authors: Armstrong Daniel W. , Nair Usha B.
Published: May 2005

Macrocyclic antibiotics are the newest class of chiral selector used in capillary electrophoresis (CE) enantioseparations. Two basic types of compounds have proven to produce useful enantioseparations. They are the ansa compounds and the glycopeptides. The ansa compounds best resolve basic or cationic racemates while the glycopeptides best resolve acidic or anionic analytes. The structure and use of these chiral selectors are discussed. The factors affecting separation and the separation mechanism are considered as well.
Analysis of antibiotics by capillary electrophoresis
Authors: Flurer Cheryl L.
Published: May 2005

The broad category of antibiotics encompasses some of the most widely prescribed pharmaceuticals in the world. As is the case with any pharmaceutical, an antibiotic must be characterized in terms of its potency or activity, and the presence and quantity of impurities. Additionally, any residue or metabolite that may be present as a result of its use must be monitored. Many capillary electrophoretic techniques have been utilized in the analysis of antibiotics, addressing the various aspects of their quantitation, profiling, and monitoring. Some of the more recent applications are summarized in this review article.
Adaptive classification of two‐dimensional gel electrophoretic spot patterns by neural networks and cluster analysis
Authors: Vohradský Jiří
Published: May 2005

The interpretation of two‐dimensional gel electrophoresis spot profiles can be facilitated by statistical and machine learning programs. Two different approaches to classification of spot profiles – cluster analysis and neural networks – are discussed. Neural networks for two different model patterns were designed and an algorithm for training of the net for the classification was developed. It was shown that the performance of neural networks is higher compared to cluster and principal component analysis. The possibility of combining both approaches into one process can increase reliability and speed of classification. Artificially created training sets with added random noise can be used for network training. The analysis was applied on the Streptomyces coelicolor developmental two‐dimensional (2‐D) gel database.
HSC‐2DPAGE and the two‐dimensional gel electrophoresis database of dog heart proteins
Authors: Dunn Michael J. , Corbett Joseph M. , Wheeler Colin H.
Published: May 2005

A two‐dimensional gel electrophoresis database of dog (Canis familiaris) proteins is presented. The database contains 1212 protein spots which have been characterised in terms of their pI and Mr. This database has been integrated into the HSC‐2DPAGE database which is accessible on the Internet via the World Wide Web with the uniform resource location (URL): (http://www.harefield.nthames.nhs.uk/nhli/protein/index.html). Identifications for 80 of the protein spots have been obtained by visual cross‐matching with the human heart protein database in HSC‐2DPAGE (42 spots), N‐terminal microsequence analysis (25 spots) and peptide mass fingerprinting (20 spots). This database is being used in studies of alterations in protein expression in models of heart failure and heart disease.
Identification and characterization of heat shock protein 27 protein species in human myocardial two‐dimensional electrophoresis patterns
Authors: Scheler Christian , Müller Eva‐Christina , Stahl Joachim , Müller‐Werdan Ursula , Salnikow Johann , Jungblut Peter
Published: May 2005

Immunostaining of heat shock protein 27 (Hsp27) protein species on two‐dimensional electrophoresis (2‐DE) gels with enhanced sensitivity yields 59 spots reacting with anti‐Hsp27 antibodies. Recombinant Hsp27 exists in 2‐DE as two major protein species which comigrate in the human myocardial pattern with Hsp27 spots C754 and D899 as defined in the heart high‐performance 2‐DE database (http://www.mdc‐berlin.de/∼emu/heart/). Preparative electrophoresis of human myocardial proteins and analysis of the enriched mass range 20–30 kDa by 2‐DE revealed eight protein spots (C438, C582, C658, C697, C754, C595, C750) from the human myocardial database and a new spot not previously detected on silver‐stained gels. These spots were identified as Hsp27 protein species by enzymatic in‐gel‐digestion and analysis by matrix assisted laser desorption‐ionization (MALDI) peptide mass fingerprinting and, in part, MALDI‐post source decay sequencing of single fragments. Possible post‐translational modifications were investigated: immunostaining tests with anti‐phospho‐serine/‐threonine/‐tyrosine antibodies, although positive for other myocardial proteins, were negative for presumed Hsp27 protein species; likewise, periodate‐glycostaining assays and biotinylation screening did not detect modifications in the investigated Hsp27 protein species.
Two‐dimensional electrophoretic analysis of myocardial proteins from lead‐exposed rabbits
Authors: Toraason Mark , Moorman William , Mathias Patty I. , Fultz Carla , Witzmann Frank
Published: May 2005

Despite reported adverse effects, the cardiovascular toxicity of lead remains controversial. The purpose of the present study was to determine if low‐level subchronic exposure of rabbits to lead would produce detectable, concentration‐dependent changes in myocardial proteins. Lead was administered to male Dutch Belted rabbits as a lead acetate solution, adjusted weekly to achieve and maintain the target blood lead levels of 0, 20, 40, and 80 μg/dL for 15 weeks. Lead exposures did not affect heart or body weights. Myocardial cocentrations of lead at sacrifice were 58 ± 25, 69 ± 23, 102 ± 62, and 105 ± 37 ng/g. Of 808 individual proteins resolved by two‐dimensional electrophoresis (2‐DE) in ventricular homogenates, 162 had coefficients of variation < 20%. A number of proteins were tentatively identified based on coordinate positions homologous to other established 2‐DE patterns. Despite variable expression of some protein spots, none of the protein abundances analyzed were found to be significantly altered (P < 0.001) by the lead exposures studied. Therefore results show no detectable effect of a low‐body burden of lead on major myocardial proteins of the rabbit.
Capillary electrophoresis analysis of isomeric truxillines and other high molecular weight impurities in illicit cocaine
Authors: Lurie Ira S. , Hays Patrick A. , Casale John F. , Moore James M. , Castell David M. , Chan King C. , Issaq Haleem J.
Published: May 2005

The analysis of by‐products and impurities in illicit cocaine, including the isomeric truxillines, is important for derivation of both strategic and tactical intelligence. In the present study, various capillary electrophoresis techniques were investigated for this purpose. The use of the anionic β‐cyclodextrin sulfobutyl ether IV as a run buffer additive at pH 8.6 gave a good separation of the truxillines and similar high molecular weight impurities in less than eight minutes. These impurities were first isolated from the bulk cocaine matrix using liquid‐liquid extraction and size‐exclusion high performance liquid chromatography. There was a red shift in the UV spectra obtained for the truxillines using photodiode array (PDA) UV detection during CE analysis. This anomalous behavior is attributed to photo‐degradation of the truxillines during the PDA‐UV irradiation process. Laser‐induced fluorescence detection using a UV krypton/fluoride laser provided greater selectivity and sensitivity versus UV detection for certain uncharacterized high molecular weight impurities.
Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis
Authors: Mansfield Elaine S. , Robertson James M. , Vainer Marina , Isenberg Alice R. , Frazier Rachael R. , Ferguson Karin , Chow ShiTse , Harris Dennis W. , Barker David L. , Gill Peter D. , Budowle Bruce , McCord Bruce R.
Published: May 2005

The profiling of polymorphic short tandem repeat (STR) markers is being applied to human identification, parentage testing and genetic mapping. Reliable genotyping of these markers is facilitated by polymerase chain reaction (PCR) amplification and high‐resolution electrophoretic separation. Capillary array electrophoresis (CAE) offers very rapid, high‐resolution separation of the amplified DNA and potential for automated sample processing not realized employing conventional slab‐gel electrophoresis. The use of CAE to type DNA samples amplified at 11 genetic loci in multiplex profiles is presented. Two sets totaling 208 samples were amplified in a multiplex fashion using AmpFlSTR‐Blue or AmpFlSTR‐Green I and analyzed in a blind study using CAE. With the exception of one sample, the CAE genotyping results were in complete agreement with results obtained using a single‐capillary system or two slab‐gel electrophoresis systems. The sample, genotype TH01 7/10, migrated similar to TH01 6.3/9.3 allele sizes, which suggested a potential band migration shift. The recommended approach to such an observation is to analyze the sample again. The sample was rerun and correct genotype verified. Allelic ladder samples were analyzed multiple times by CAE to determine sizing accuracy and precision. The sizing of over 240 allelic ladder samples vielded an average within‐run precision of ± 0.13 bp and between‐run precision of ± 0.21 bp for fragments up to 350 bp. The CAE protocols permit processing of up to 96 multiplex STR samples in under 70 min.
Electrophoretic applications of phycobiliproteins
Authors: Aráoz Rómulo , Lebert Michael , Häder Donat‐P.
Published: May 2005

Phycobiliproteins are homologous chromoproteins which constitute the phycobilisomes, the light harvesting complexes of the photosynthetic apparatus in cyanobacteria, rhodophyta and cryptophyta. In the present work, phycocyanin (PC) and Phycoerythrin (PE) from a Nostoc species are proposed as protein markers for electrophoretic techniques. Phycocyanin is a blue‐colored phycobiliprotein; it carries phycocyanobilin as chromophoric group and is composed of two subunits, α and β, with Mr of 14000 and 17000, respectively. In contrast, the PE subunits, having a similar Mr of 21000, are deep rose chromoproteins and carry phycoerythrobilin residues. Both low molecular weight phycobiliproteins are also suitable for monitoring protien blotting and the focusing time of protein samples during isoelectric focusing as internal markers. The PE subunits which form a single broad band after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis have different isoelectric points, and they form two visible bands when they reach their isoelectric point. The phycobilisomes constitute up to 50% of the total protein in cyanobacteria and their content in PC or PE can be up‐ or down‐regulated by using different light conditions (chromatic adaptation).
Double enzyme‐catalyzed microreactors using capillary electrophoresis
Authors: Zhao Dong S. , Gomez Frank A.
Published: May 2005

This work evaluates the concept of a double enzyme‐catalyzed microreactor using capillary electrophoresis (CE). Migrating in a capillary under electrophoresis conditions, plugs of substrate and two enzymes are injected separately in buffer and allowed to react. Extent of reaction and product ratios were subsequently determined by CE. This concept is demonstrated using two model systems: the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) by hexokinase (HK, EC 2.7.1.1) and apyrase (APY, EC 3.6.1.5), respectively, in the conversion of glucose to glucose‐6‐phosphate and inorganic phosphate, respectively, and the conversion of nicotinamide adenine dinucleotide, reduced form (NADH), to nicotinamide adenine dinucleotide (NAD) and back to NADH by lactate dehydrogenase (LDH, EC 1.1.1.27) and glucose‐6‐phosphate dehydrogenase (G6PDH, EC 1.1.1.49), respectively, in the conversion of pyruvate to lactate and glucose‐6‐phosphate (glc‐6‐P) to 6‐phosphogluconate, respectively. These procedures illustrate the use of the capillary as a double microreactor and the ease of quantitation of reaction products under conditions of electrophoresis.
Whole genome scan for habitat‐specific genes by signature‐tagged mutagenesis (minireview)
Authors: Hensel Michael
Published: May 2005

Large numbers of new open reading frames can be identified by whole genome sequencing of microbial genomes. Efficient new approaches are required to investigate the role of the putative genes, or to identify genes required in distinct habitats. The novel technique ‘signature‐tagged mutagenesis’ allows the identification of individual mutants within complex pools of mutants. Large numbers of mutants can be analyzed in a parallel manner for negative phenotypes like loss of function or attenuation of virulence. Further analysis of mutations identified by a negative selection procedure can be performed by linking the position of the respective mutations to genes identified by whole genome sequencing of microbes.
A peptide concentration and purification method for protein characterization in the subpicomole range using matrix assisted laser desorption/ionization‐postsource decay (MALDI‐PSD) sequencing
Authors: Gevaert Kris , Demol Hans , Sklyarova Tatyana , Vandekerckhove Joël , Houthaeve Tony
Published: May 2005

We here describe the use of added reversed‐phase chromatographic beads to concentrate peptides from highly diluted solutions. In the procedure developed, peptide‐bead suspensions are dried under vacuum to complete dryness; peptides are subsequently eluted in a small volume of matrix‐assisted laser desorption/ionization (MALDI)‐matrix containing organic/aqueous solvent and transferred to a MALDI‐target for mass analysis. We show that by using this bead‐peptide concentration procedure, low femtomole amounts of peptides are efficiently concentrated, up to 1000 times, to volumes smaller than 0.7 μuL. We have used this concentration procedure in combination with MALDI‐post‐source decay analysis to identify subpicomole amounts of proteins present in polyacrylamide gels. Furthermore, we show that the bead‐peptide concentration method can be elegantly used to clean up samples contaminated with high concentrations of substances normally deleterious to MALDI‐mass spectrometry (MS) experiments. We have found additionally that the bead‐peptide concentration procedure can be successfully used to store low femtomole amounts of peptide for prolonged periods of time without severe losses of peptide material. This bead‐peptide concentration procedure therefore seems to be a simple and convenient step in the MALDI‐MS sample preparation process.
Use of liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS/MS) for routine identification of enzymatically digested proteins separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Stone Kathryn L. , Deangelis Raymond , LoPresti Mary , Jones Joshua , Papov Vladimir V. , Williams Kenneth R.
Published: May 2005

Automated liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS/MS) analysis of >100 tryptic digests carried out on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) separated, Coomassie Blue‐stained proteins that were prepared by >50 different laboratories demonstrates that a commercial electrospray/quadrupole ion trap mass spectrometer and the tandem mass correlation algorithm developed by Eng et al. (Am. Soc. Mass Spectrom. 1994, 5, 976–989) provide an extremely robust and facile approach to routine protein identification. By requiring a minimum of two significant matches to peptides that would be predicted to be produced by the protease that was used, low pmol levels of proteins can be identified with high confidence while minimizing the probability of identifying the protease itself and/or the ubiquitous contaminant, keratin. Hence, in only 7% of the digests analyzed was keratin identified and in only 5% of the digests analyzed was the protease itself identified. In contrast, 58% of the analyzed samples were identified and, in many instances, multiple proteins were identified in the same sample. Although the median amount of digest analyzed was 6.1 pmol, the limit of sensitivity (as the instrument is configured with a flow rate of 4 μL/min) appears to be at the 500 fmol level. Since one of the primary reasons for not identifying a sample is that its sequence is not yet in the database searched, the utility of an LC MS/MS approach to protein identification will certainly increase in the future as the sequences of more genomes are completed.
High‐resolution density gradient electrophoresis of subcellular organelles and proteins under nondenaturing conditions
Authors: Tulp Abraham , Fernandez‐Borja Mar , Verwoerd Desirée , Neefjes Jacques
Published: May 2005

We have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high‐resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis 1993, 14, 1011–1018). Isoforms of bovine β‐lactoglobulin, human apo‐transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and proteins.
Natural resistance to intracellular parasites: A study by two‐dimensional gel electrophoresis coupled with multivariate analysis
Authors: Kovářová Hana , Radzioch Danuta , Hajdúch Marián , Šírová Milada , Bláha Václav , Macela Aleš , Stulík Jiří , Hernychová Lenka
Published: May 2005

Natural resistance to Mycobacterium bovis bacillus Calmette‐Guérin (BCG) is determined by the Bcg gene (Nramp1), which is exclusively expressed by mature macrophages. The Nramp1 gene is a dominant autosomal gene that has two allelic forms; r confers resistance and s confers susceptibility to infection with intracellular pathogen. Although the wide range of pleiotropic immunological effects of the Nramp1 gene has been described, the exact mechanism of its action remains elusive. In this study we searched for differentially expressed proteins that might provide clues in the studies on Nramp1 gene function. We performed two‐dimensional gel electrophoresis of cellular proteins prepared from a B10R macrophage line derived from mice carrying the r allele of the Nramp1 gene, B10S macrophages carrying the s allele, and B10R‐Rb macrophages transfected with Nrampl‐ribozyme. The classification of protein patterns and selection of distinct proteins characteristic of r or s allele‐carrying macrophages was performed using the principal component analysis. We found differential expression of four proteins with the following isoelectric point/molecular weight (pI/Mr) in B10R macrophages compared to B10S and B10R‐Rb macrophages: 6.6/25, 7.0/22, 9.1/31.5, and 5.3/8.5. The protein 7.0/22 has been identified as Mn‐superoxide dismutase and the best candidate for protein p6.6/25 seems to be Bcl‐2 according to the immunoblot analysis. When the splenic macrophages carring the r or s allele were analyzed, the changes in relative abundance for proteins 6.6/25 and p7.0/22 were satisfactorily reproduced. Overall, the two identified proteins are important in the regulation of intracellular redox balance and the regulation of apoptosis in macrophages, respectively. Our findings may suggest their possible biological role in the innate immunity against intracellular pathogens.
Amplification of microsatellites adapted from human systems in faecal DNA of wild Hanuman langurs (Presbytis entellus)
Authors: Launhardt Kristin , Epplen Cornelia , Epplen Jörg T. , Winkler Paul
Published: May 2005

Microsatellite systems originally established for human DNA were utilized for paternity testing from faecal DNA in a natural population of Hanuman langurs (Presbytis entellus). Thirty‐two primer pairs were applied to amplify DNA obtained from langur faeces. Twenty‐two of these primer pairs yielded specific amplification products and 11 loci were polymorphic. Allele distributions and heterozygosity rates were determined for five systems. Genetic information from these five systems was sufficient for paternity exclusion in 46 out of 52 cases. Results were consistent enough to allow genotyping from faeces, although sometimes only one allele was amplified in a heterozygous individual. In conclusion, relationship analyses from faeces are possible in spite of the evolutionary distance between humans and langurs.
The effect of complexation additives on analyte migration behavior in capillary electrochromatography
Authors: Bowser Michael T. , Chen David D. Y.
Published: May 2005

In capillary electrochromatography (CEC), analytes often have different mobilities in the mobile phase, and often are involved in multiple equilibria. In this paper, the migration behavior of an analyte in CEC is described by a general equation in which individual capacity factors are used to describe the tendency of the analyte to exist as the various analyte species present in a separation system, and the effects of both field and equilibrium are accounted for. The resolution of two analytes is shown to be related linearly to the ratio of their migration rates. The effect of the electroosmotic flow (EOF) in CEC is more complicated than in CE because it is experienced only by a fraction of the analyte, whereas in CE, it is experienced by all analyte species. A procedure for calculating the electrophoretic mobility of the analyte based on the fraction of the analyte in the buffer is demonstrated. The effect of the EOF on resolution is also discussed.
Two‐dimensional electrophoresis of proteins in an immobilized pH 4–12 gradient
Authors: Görg Angelika , Boguth Günther , Obermaier Christian , Weiss Walter
Published: May 2005

For checking theoretical two‐dimensional (2‐D) maps derived from sequenced genomes, indicating that nonnegligible amounts of proteins up to pH 12 are to be expected, a wide‐range immobilized pH 4–12 gradient was generated. Depending on the extraction method of sample preparation, proteins with pIs up to pH 12 are detected in a single gel. Highly reproducible protein patterns focused to the steady state with round‐shaped spots up to pH 12 are obtained with the standard protocol originally described in 1988 (Görg et al., Electrophoresis 1988, 9, 531–546).
Recent developments in DNA electrophoretic separations
Authors: Slater Gary W. , Kist Tarso B. L. , Ren Hongji , Drouin Guy
Published: May 2005

DNA electrophoresis is now a fairly mature technology. Nevertheless, as we approach the 21st century, new ideas are frequently suggested that could lead to a revolution for DNA sequencing and mapping. Here, we review some of the novel concepts that have been studied since ca. 1990. Our review focuses on new separation mechanisms, new sieving matrices and recent conceptual advances.
Human globin chain separation by capillary electrophoresis in acidic isoelectric buffers
Authors: Righetti Pier Giorgio , Saccomani Arianna , Stoyanov Alexander V. , Gelfi Cecilia
Published: May 2005

A simple and reliable method, utilizing capillary electrophoresis in uncoated capillaries in acidic isoelectric buffers, is reported for screening for thalassemia and other defects on the synthesis of human globin chains. A solution of 50 mM iminodiacetic acid (pi2.23), containing 7 M urea and 0.5% hydroxyethyl‐cellulose (apparent pH 3.2) is used as background electrolyte for fast separation of heme‐free, denatured globin (α, β and γ) chains. Due to the low conductivity of such a buffer, high voltage gradients (600 V/cm) can be applied, thus reducing the separation time to only a few minutes. It is additionally shown that inclusion of 2% surfactant (Tween 20) in the background electrolyte induces the splitting of the γ chains into two zones, called Aγ and Gγ, which represent the products of two genes coding for Ala or Gly as residue 136 of the chain.
Electrophoresis combined with novel mass spectrometry techniques: Powerful tools for the analysis of proteins and proteomes
Authors: Figeys Daniel , Gygi Steven P. , Zhang Yanni , Watts Julian , Gu Ming , Aebersold Ruedi
Published: May 2005

Analytical and preparative electrophoresis separation techniques have been essential tools in protein biochemistry and the biological sciences in general. The combination of high resolution electrophoresis techniques with high performance analytical procedures has dramatically enhanced analytical protein biochemistry. In this report we describe the combination of electrophoretic separation techniques with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). A series of different techniques, consisting of automated high performance liquid chromatography (HPLC)‐MS/MS, capillary‐HPLC‐MS/MS, and solid phase extraction (SPE)‐capillary zone electrophoresis (CZE)‐MS/MS, are described in the context of the identification of high pmol to the low fmol amounts of proteins. Application of these powerful new tools for the analysis of proteins on a large proteome‐wide scale is presented. Furthermore, the combination of orthogonal separation techniques, such as immobilized metal affinity chromatography (IMAC) with SPE‐CZE‐MS/MS, and IMAC followed by HPLC, and by SPE‐CZE‐MS/MS, are presented for the detailed investigation of post‐translational modifications of specific proteins.
Reference map of the low molecular mass proteins of Haemophilus influenzae
Authors: Fountoulakis Michael , Juranville Jean‐François , Röder Daniel , Evers Stefan , Berndt Peter , Langen Hanno
Published: May 2005

Analysis of the proteome of Haemophilus influenzae by two‐dimensional polyacrylamide gel electrophoresis on conventional Tris‐glycine gels does not usually result in efficient separation of the proteins in the 5–20 kDa range, which are mainly accumulated in the lower acidic and basic regions. In order to improve the separation of the low molecular mass proteins, we used homogeneous Tricine gels of two urea concentrations in the second‐dimensional separation. The Tricine gel systems allowed the efficient and reproducible separation of the proteins of the microorganism with masses between 5 and 20 kDa, however, no proteins with masses below 5 kDa could be visualized. Approximately 80 proteins migrating in the 5–25 kDa region were identified by matrix assisted laser desorption/ionization ‐ mass spectrometry, of which 40 identified for the first time. The digestion of the low mass proteins often produced only few peptides, which were insufficient for confident identification by mass spectrometry. Therefore, the identification was occasionally achieved by a sequential digestion with two proteases, trypsin or endoproteinase Lys‐C as first and carboxypeptidase P as second enzyme. The gel system described may be useful for the efficient separation of low molecular mass proteins from other organisms to construct standard maps.
Capillary electrochromatography of derivatized mono‐ and oligosaccharides
Authors: Yang Changming , Rassi Ziad El
Published: May 2005

An octadecyl‐silica (ODS) stationary phase with light surface coverage of octadecyl ligands was introduced for capillary electrochromatography (CEC) at moderate electroosmotic flow (EOF) velocity. The ODS stationary phase was intentionally produced with light surface coverage in order to ensure a moderate EOF velocity across the packed capillary column, thus allowing relatively rapid analysis time. Despite the fact that the stationary phase leaves 75% of the surface silanols unreacted, fused‐silica capillary columns packed with this ODS stationary phase exhibited reversed‐phase behavior toward neutral alkylbenzene homologous solutes using hydroorganic eluents. Closely related p‐nitrophenylglycosides including some p‐nitrophenyl‐monosaccharides and p‐nitrophenyl‐maltooligosaccharides were readily separated on the ODS capillary column within a relatively short analysis time. Also, α‐ and β‐anomers of some p‐nitrophenyl‐monosaccharides were readily separated in the presence of a small amount of borate buffer in the hydroorganic eluent.
Capillary electrochromatography with novel stationary phases. I. Preparation and characterization of octadecyl‐sulfonated silica
Authors: Zhang Minquan , Rassi Ziad El
Published: May 2005

A novel silica‐based stationary phase was developed for use in capillary electrochromatography (CEC) at relatively high electroosmotic flow (EOF). The silica was first bonded with a relatively hydrophilic layer bearing strong sulfonic acid groups. To this charged polar sublayer, octadecyl functions were covalently attached to yield the nonpolar top layer. This novel stationary phase, referred to as octadecylsulfonated silica (ODSS), was packed in bare fusedsilica capillaries or in capillaries with the same coating as the sublayer on the silica‐based stationary phase. The resulting packed columns were evaluated in CEC using alkylbenzenes as the test model solutes. Good separations can be achieved in less than 8 min, much faster than when using a regular octadecyl silica capillary column. Due to the permanent negative charge provided by the sulfonated sublayer on both the capillary walls and the silica particles, the magnitude of the EOF remained more or less constant over a wide range of pH, and its magnitude can be conveniently varied by the applied voltage.
Pressure‐assisted and pressure‐programmed capillary electrophoresis/electrospray ionization time of flight ‐ mass spectrometry for the analysis of peptide mixtures
Authors: Cao Ping , Moini Mehdi
Published: May 2005

Pressure assisting and pressure programming the inlet of the capillary electrophoresis instrument were used for the analysis of peptide mixtures and protein digests using capillary electrophoresis/electrospray ionization ‐ mass spectrometry (CE/ESI‐MS). CE/ESI‐MS of peptide mixtures and tryptic digests of proteins was studied using three different types of capillary columns: (i) a freshly aminopropylsilane (APS)‐treated column, (ii) an untreated column, and (iii) a degraded APS‐treated column. To maintain a constant and adequate buffer flow toward the CE capillary outlet for stable CE and ESI operation, low pressure was applied to the inlet of the CE when an untreated or degraded APS capillary was used. By programming the inlet pressure, CE/ESI‐MS analysis time was reduced to 1/3 of its original time. The utility of this technique is demonstrated by CE/ESI‐MS analysis of a hemoglobin variant (hemoglobin‐S) and its tryptic digests. Identification of the mutant peptide in the tryptic digest of hemoglobin‐S was achieved by collision‐induced dissociation (CID) of the protein digests using CE/ESI time of flight ‐ mass spectrometry (TOF‐MS).
Capillary zone electrophoresis of proteins with fused‐silica capillaries having polymers and surfactants adsorbed onto surfactant moieties previously covalently bound to the capillary column surface
Authors: Yang Changming , Rassi Ziad El
Published: May 2005

Fused‐silica capillary columns having cationic surfactant moieties (CSM) covalently attached to the capillary inner walls were introduced for the separation of proteins by capillary zone electrophoresis (CZE). The CSM capillary coating proved to be useful in the separation of basic and acidic proteins when modified hydroxypropylcellulose (HPC), namely epoxybutane‐HPC (EBHPC), was adsorbed to the primary CSM coating, yielding a hybrid coating (i.e., coating consisting of a covalenty bound ligand and an adsorbed ligand). The EBHPC layer rendered the capillary surface highly hydrophilic, thus permitting the separation of basic proteins with relatively high plate counts. Also, the CSM coating was useful for the separation of acidic proteins when the capillary wall had, in addition, a negatively charged polymer adsorbed to the surface, e.g., hyaluronic acid. In general, neutral and charged polymeric compounds could be readily adsorbed by the CSM coating, thus altering the zeta potential of the capillary and diminishing solute adsorption to the capillary surface. In other words, the sign of the zeta potential of the capillary surface could be tailored to be of the same sign as the charge of the analytes. Under these conditions, little or no solute‐wall interaction could be observed due to electrostatic repulsion. Although the capillary surface was charged, the presence of adsorbed polymer suppressed or, in most cases, even eliminated the electroosmotic flow (EOF).
Comparison of protein mixtures in aqueous humor by membrane preconcentration ‐ capillary electrophoresis ‐ mass spectrometry
Authors: Rohde E. , Tomlinson Andy J. , Johnson D. H. , Naylor Stephen
Published: May 2005

The significance of proteomic research is coupled with the recent exponential growth of these investigations. Currently, the most popular techniques used for these studies include the coupling of 1‐ and 2‐dimensional electrophoresis with mass spectrometric analysis of the extracted and digested proteins. However, detection limits of gel staining methods have led to a search for complimentary techniques that afford the detection of lower concentrations of biologically relevant proteins. In the present studies, we have evaluated the applicability of on‐line capillary electrophoresis — mass spectrometry (CE‐MS) for this application. Specifically, we used membrane preconcentration‐CE‐MS (mPC‐CE‐MS) to analyze 13 samples of human aqueous humor (AH) from patients with various ocular pathologies (cataract, cataract plus glaucoma, and cataract plus pseudoexfoliation syndrome). This approach enabled rapid analysis of a relatively large volume (1) μL of each specimen, and a protein map for each was created. Measured average molecular weights (Mr) were used to tentatively identify proteins after search of the SWISS‐PROT database using Tagldent from ExPaSy. Among those proteins tentatively identified are β‐2 microglobulin (Mr 11731.2), apolipoprotein A1 (Mr 28078.6) and serum albumin (Mr 66400). Proteins with Mr of 4349 (unidentified), 11731.2 (β‐2 microglobulin), 13400–14100 (immunoglobulin fragments), 28078.2 (apolipoprotein A1) and ∼68000 (serum albumin) were observed in the majority of specimens. Generally no significant differences were noted in the protein composition of aqueous humor samples from different pathologies. However, the absence of an Mr 13345 protein and its oxidized form (Mr 13361) in samples from patients with pseudoexfoliation syndrome was noted. Occasionally the α‐and β‐chains of hemoglobin, a contaminant in aqueous humor introduced during sampling, were also detected. We conclude from these studies that mPC‐CE‐MS is an attractive complimentary technique for proteome research, as this approach enables direct mapping and characterization of low concentrations of proteins that are present in complex physiologically derived fluids.