Abstract:
A method of mass spectrometry is disclosed wherein a gas collision cell is repeatedly switched between a fragmentation and a non-fragmentation mode. Parent ions from a first sample are passed through the collision cell and parent ion mass spectra and fragmentation ion mass spectra are obtained. Parent ions from a second sample are then passed through the collision cell and a second set of parent ion mass spectra and fragmentation ion mass spectra are obtained. The mass spectra are then compared and if either certain parent ions or certain fragmentation ions in the two samples are expressed differently then further analysis is performed to seek to identify the ions which are expressed differently in the two different samples.

Description:
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
         [0001]    A preferred embodiment will now be described with reference to FIG. 1. A mass spectrometer  6  is shown which comprises an ion source  1 , preferably an Electrospray lonisation source, an ion guide  2 , a quadrupole mass filter  3 , a collision cell or other fragmentation device  4  and an orthogonal acceleration Time of Flight mass analyser  5  incorporating a reflectron. The ion guide  2  and mass filter  3  may be omitted if necessary. The mass spectrometer  6  is preferably interfaced with a chromatograph, such as a liquid chromatograph (not shown) so that the sample entering the ion source  1  may be taken from the eluent of the liquid chromatograph.  
           [0002]    The quadrupole mass filter  3  is disposed in an evacuated chamber which is maintained at a relatively low pressure e.g. less than 10 B5  10 −5  mbar. The rod electrodes comprising the mass filter  3  are connected to a power supply which generates both RF and DC potentials which determine the mass to charge value transmission window of the mass filter  3 .  
           [0003]    The collision cell  4  preferably comprises either a quadrupole or hexapole rod set which may be enclosed in a substantially gas-tight casing (other than having a small ion entrance and exit orifice) into which a collision gas such as helium, argon, nitrogen, air or methane may be introduced at a pressure of between 10 −4  and 10 −1  mbar, further preferably 10 −3  mbar to 10 −2  mbar. Suitable AC or RF potentials for the electrodes comprising the collision cell  4  are provided by a power supply (not shown).  
           [0004]    Ions generated by the ion source  1  are transmitted by ion guide  2  and pass via an interchamber orifice  7  into vacuum chamber  8 . Ion guide  2  is maintained at a pressure intermediate that of the ion source and the vacuum chamber  8 . In the embodiment shown, ions are mass filtered by mass filter  3  before entering collision cell  4 . However, the mass filter  3  is an optional feature of this embodiment. Ions exiting from the collision cell  4  pass into a Time of Flight mass analyser  5 . Other ion optical components, such as further ion guides and/or electrostatic lenses, may be provided which are not shown in the figures or described herein. Such components may be used to maximise ion transmission between various parts or stages of the apparatus. Various vacuum pumps (not shown) may be provided for maintaining optimal vacuum conditions. The Time of Flight mass analyser  5  incorporating a reflectron operates in a known way by measuring the transit time of the ions comprised in a packet of ions so that their mass to charge ratios can be determined.  
           [0005]    A control means (not shown) provides control signals for the various power supplies (not shown) which respectively provide the necessary operating potentials for the ion source  1 , ion guide  2 , quadrupole mass filter  3 , collision cell  4  and the Time of Flight mass analyser  5 . These control signals determine the operating parameters of the instrument, for example the mass to charge ratios transmitted through the mass filter  3  and the operation of the analyser  5 . The control means may be a computer (not shown) which may also be used to process the mass spectral data acquired. The computer can also display and store mass spectra produced by the analyser  5  and receive and process commands from an operator. The control means may be automatically set to perform various methods and make various determinations without operator intervention, or may optionally require operator input at various stages.  
           [0006]    The control means is also preferably arranged to switch the collision cell or other fragmentation device  4  back and forth repeatedly and/or regularly between at least two different modes. In one mode a relatively high voltage such as greater than or equal to 15V is applied to the collision cell  4  which in combination with the effect of various other ion optical devices upstream of the collision cell  4  is sufficient to cause a fair degree of fragmentation of ions passing therethrough. In a second mode a relatively low voltage such as less than or equal to 5V is applied which causes relatively little (if any) significant fragmentation of ions passing therethrough.  
           [0007]    In one embodiment the control means may switch between modes approximately every second. When the mass spectrometer  6  is used in conjunction with an ion source  1  being provided with an eluent separated from a mixture by means of liquid or gas chromatography, the mass spectrometer  6  may be run for several tens of minutes over which period of time several hundred high and low fragmentation mass spectra may be obtained.  
           [0008]    At the end of the experimental run the data which has been obtained is analysed and parent ions and fragment ions can be recognised on the basis of the relative intensity of a peak in a mass spectrum obtained when the collision cell  4  was in one mode compared with the intensity of the same peak in a mass spectrum obtained approximately a second later in time when the collision cell  4  was in the second mode.  
           [0009]    According to an embodiment, mass chromatograms for each parent and fragment ion are generated and fragment ions are assigned to parent ions on the basis of their relative elution times. 
       
    
    
       [0010]    An advantage of this method is that since all the data is acquired and subsequently processed then all fragment ions may be associated with a parent ion by closeness of fit of their respective elution times. This allows all the parent ions to be identified from their fragment ions, irrespective of whether or not they have been discovered by the presence of a characteristic fragment ion or characteristic “neutral loss”.  
         [0011]    According to another embodiment an attempt is made to reduce the number of parent ions of interest. A list of possible (i.e. not yet finalised) parent ions of interest may be formed by looking for parent ions which may have given rise to a predetermined fragment ion of interest e.g. an immonium ion from a peptide. Alternatively, a search may be made for parent and fragment ions wherein the parent ion could have fragmented into a first component comprising a predetermined ion or neutral particle and a second component comprising a fragment ion. Various steps may then be taken to further reduce/refine the list of possible parent ions of interest to leave a number of parent ions of interest which are then preferably subsequently identified by comparing elution times of the parent ions of interest and fragment ions. As will be appreciated, two ions could have similar mass to charge ratios but different chemical structures and hence would most likely fragment differently enabling a parent ion to be identified on the basis of a fragment ion.  
         [0012]    A sample introduction system is shown in more detail in FIG. 2. Samples may be introduced into the mass spectrometer  6  by means of a Micromass (RTM) modular CapLC system. For example, samples may be loaded onto a C18 cartridge (0.3 mm×5 mm) and desalted with 0.1% HCOOH for 3 minutes at a flow rate of 30_L 30 μL per minute. A ten port valve may then switched such that the peptides are eluted onto the analytical column for separation, see inset of FIG. 2. Flow from two pumps A and B may be split to produce a flow rate through the column of approximately 200 nl/min.  
         [0013]    A preferred analytical column is a PicoFrit (RTM) column packed with Waters (RTM) Symmetry C18 set up to spray directly into the mass spectrometer  6 . An electrospray potential (ca. 3 kV) may be applied to the liquid via a low dead volume stainless steel union. A small amount e.g. 5 psi (34.48 kPa) of nebulising gas may be introduced around the spray tip to aid the electrospray process.  
         [0014]    Data can be acquired using a mass spectrometer  6  fitted with a Z-spray (RTM) nanoflow electrospray ion source. The mass spectrometer may be operated in the positive ion mode with a source temperature of 80° C. and a cone gas flow rate of 401/hr.  
         [0015]    The instrument may be calibrated with a multi-point calibration using selected fragment ions that result, for example, from the collision-induced decomposition (CID) of Glu-fibrinopeptide b. Data may be processed using the MassLynx (RTM) suite of software.  
         [0016]    [0016]FIGS. 3A and 3B show respectively fragment and parent ion spectra of a tryptic digest of alcohol dehydrogenase (ADH). The fragment ion spectrum shown in FIG. 3A was obtained while the collision cell voltage was high, e.g. around 30V, which resulted in significant fragmentation of ions passing therethrough. The parent ion spectrum shown in FIG. 3B was obtained at low collision energy e.g. less than or equal to 5V. The data presented in FIG. 3B was obtained using a mass filter  3  upstream of collision cell  4  and set to transmit ions having a mass to charge value greater than 350. The mass spectra in this particular example were obtained from a sample eluting from a liquid chromatograph, and the spectra were obtained sufficiently rapidly and close together in time that they essentially correspond to the same component or components eluting from the liquid chromatograph.  
         [0017]    In FIG. 3B, there are several high intensity peaks in the parent ion spectrum, e.g. the peaks at 418.7724 and 568.7813, which are substantially less intense in the corresponding fragment ion spectrum shown in FIG. 3A. These peaks may therefore be recognised as being parent ions. Likewise, ions which are more intense in the fragment ion spectrum shown in FIG. 3A than in the parent ion spectrum shown in FIG. 3B may be recognised as being fragment ions. As will also be apparent, all the ions having a mass to charge value less than 350 in the high fragmentation mass spectrum shown in FIG. 3A can be readily recognised as being fragment ions on the basis that they have a mass to charge value less than 350 and the fact that only parent ions having a mass to charge value greater than 350 were transmitted by the mass filter  5  to the collision cell  4 .  
         [0018]    FIGS.  4 A-E show respectively mass chromatograms for three parent ions and two fragment ions. The parent ions were determined to have mass to charge ratios of 406.2 (peak “MCI”), 418.7 (peak “MC 2 ”) and 568.8 (peak “MC 3 ”) and the two fragment ions were determined to have mass to charge ratios of 136.1 (peaks “MC 4 ” and “MC 5 ”) and 120.1 (peak “MC 6 ”).  
         [0019]    It can be seen that parent ion peak MC 1  (m/z 406.2) correlates well with fragment ion peak MC 5  (m/z 136.1) i.e. a parent ion with a mass to charge ratio of 406.2 seems to have fragmented to produce a fragment ion with a mass to charge ratio of 136.1. Similarly, parent ion peaks MC 2  and MC 3  correlate well with fragment ion peaks MC 4  and MC 6 , but it is difficult to determine which parent ion corresponds with which fragment ion.  
         [0020]    [0020]FIG. 5 shows the peaks of FIGS.  4 -E overlaid on top of one other and redrawn at a different scale. By careful comparison of the peaks of MC 2 , MC 3 , MC 4  and MC 6  it can be seen that in fact parent ion MC 2  and fragment ion MC 4  correlate well whereas parent ion MC 3  correlates well with fragment ion MC 6 . This suggests that parent ions with a mass to charge ratio of 418.7 fragmented to produce fragment ions with a mass to charge ratio of 136.1 and that parent ions with mass to charge ratio 568.8 fragmented to produce fragment ions with a mass to charge ratio of 120.1.  
         [0021]    This cross-correlation of mass chromatograms may be carried out using automatic peak comparison means such as a suitable peak comparison software program running on a suitable computer.  
         [0022]    [0022]FIG. 6 show the mass chromatogram for the fragment ion having a mass to charge ratio of 87.04 extracted from a HPLC separation and mass analysis obtained using mass spectrometer  6 . It is known that the immonium ion for the amino acid Asparagine has a mass to charge value of 87.04. This chromatogram was extracted from all the high energy spectra recorded on the mass spectrometer  6 . FIG. 7 shows the full mass spectrum corresponding to scan number  604 . This was a low energy mass spectrum recorded on the mass spectrometer  6 , and is the low energy spectrum next to the high energy spectrum at scan  605  that corresponds to the largest peak in the mass chromatogram of mass to charge ratio 87.04. This shows that the parent ion for the Asparagine immonium ion at mass to charge ratio 87.04 has a mass of 1012.54 since it shows the singly charged (M+H) +  ion at mass to charge ratio 1013.54, and the doubly charged (M+2H) ++  ion at mass to charge ratio 507.27.  
         [0023]    [0023]FIG. 8 shows a mass spectrum from the low energy spectra recorded on mass spectrometer  6  of a tryptic digest of the protein_Casein β-Casein. The protein digest products were separated by HPLC and mass analysed. The mass spectra were recorded on the mass spectrometer  6  operating in the MS mode and alternating between low and high collision energy in the gas collision cell  4  for successive spectra. FIG. 9 shows a mass spectrum from the high energy spectra recorded at substantially the same time that the low energy mass spectrum shown in FIG. 8 relates to. FIG. 10 shows a processed and expanded view of the mass spectrum shown in FIG. 9 above. For this spectrum, the continuum data has been processed so as to identify peaks and display them as lines with heights proportional to the peak area, and annotated with masses corresponding to their centroided masses. The peak at mass to charge ratio 1031.4395 is the doubly charged (M+2H) ++  ion of a peptide, and the peak at mass to charge ratio 982.4515 is a doubly charged fragment ion. It has to be a fragment ion since it is not present in the low energy spectrum. The mass difference between these ions is 48.9880. The theoretical mass for H 3 PO 4  is 97.9769, and the mass to charge value for the doubly charged H 3 PO 4   ++  ion is 48.9884, a difference of only 8 ppm from that observed. It is therefore assumed that the peak having a mass to charge ratio of 982.4515 relates to a fragment ion resulting from a peptide ion having a mass to charge of 1031.4395 losing a H 3 PO 4   ++  ion.  
         [0024]    Some experimental data is now presented which illustrates the ability of the preferred embodiment to quantify the relative abundance of two proteins contained in two different samples which comprise a mixture of proteins.  
         [0025]    A first sample contained the tryptic digest products of three proteins BSA, Glycogen Phosphorylase B and Casein. These three proteins were initially present in the ratio 1:1:1. Each of the three proteins had a concentration of 330 fmol/ — 1 fmol/μl. A second sample contained the tryptic digest products of the same three proteins BSA, Glycogen Phosphorylase B and Casein. However, the proteins were initially present in the ratio 1:1:X. X was uncertain but believed to be in the range 2-3. The concentration of the proteins BSA and Glycogen Phosphorylase B in the second sample mixture was the same as in the first sample, namely 330 fmol/ — 1 fmol/μl.  
         [0026]    The experimental protocol which was followed was that 1 — 1 of sample was loaded for separation on to a HPLC column at a flow rate of 4 — 1/min 4 μl/min. The liquid flow was then split such that the flow rate to the nano-electrospray ionisation source was approximately 200 nl/min.  
         [0027]    Mass spectra were recorded on the mass spectrometer  6 . Mass spectra were recorded at alternating low and high collision energy using nitrogen collision gas. The low-collision energy mass spectra were recorded at a collision voltage of 10V and the high-collision energy mass spectra were recorded at a collision voltage of 33V. The mass spectrometer was fitted with a Nano-Lock-Spray device which delivered a separate liquid flow to the source which may be occasionally sampled to provide a reference mass from which the mass calibration may be periodically validated. This ensured that the mass measurements were accurate to within an RMS accuracy of 5 ppm. Data were recorded and processed using the MassLynx (RTM) data system.  
         [0028]    The first sample was initially analysed and the data was used as a reference. The first sample was then analysed a further two times. The second sample was analysed twice. The data from these analyses were used to attempt to quantify the (unknown) relative abundance of Casein in the second sample.  
         [0029]    All data files were processed automatically generating a list of ions with associated areas and high-collision energy spectra for each experiment. This list was then searched against the Swiss-Prot protein database using the ProteinLynx (RTM) search engine. Chromatographic peak areas were obtained using the Waters (RTM) Apex Peak Tracking algorithm. Chromatograms for each charge state found to be present were summed prior to integration.  
         [0030]    The experimentally determined relative expression level of various peptide ions normalised with respect to the reference data for the two samples are given in the following tables.  
                                                                                             Sample 1   Sample 1   Sample 2   Sample 2               Run 1   Run 2   Run 1   Run 2                                    BSA peptide ions                               FKDLGEEHFK   (SEQ ID NO: 1)   0.652   0.433   0.914   0.661               HLVDEPQNLIK   (SEQ ID NO: 2)   0.905   0.829   0.641   0.519               KVPQVSTPTLVEVSR   (SEQ ID NO: 3)   1.162   0.787   0.629   0.635               LVNELTEFAK   (SEQ ID NO: 4)   1.049   0.795   0.705   0.813               LGEYGFQNALIVR   (SEQ ID NO: 5)   1.278   0.818   0.753   0.753               AEFVEVTK   (SEQ ID NO: 6)   1.120   0.821   0.834   0.711               Average       1.028   0.747   0.746   0.682               Glycogen       Phophorylase B       Peptide ions       VLVDLER   (SEQ ID NO: 7)   1.279   0.751   n/a   0.701               TNFDAFPDK   (SEQ ID NO: 8)   0.798   0.972   0.691   0.699               EIWGVEPSR   (SEQ ID NO: 9)   0.734   0.984   1.053   1.054               LITAIGDVVNHDPVVGDR   (SEQ ID NO: 10)   1.043   0.704   0.833   0.833               VLPNDNFFEGK   (SEQ ID NO: 11)   0.969   0.864   0.933   0.808               QIIEQLSSGFFSPK   (SEQ ID NO: 12)   0.691   n/a   1.428   1.428               VAAAFPGDVDR   (SEQ ID NO: 13)   1.140   0.739   0.631   0.641               Average       0.951   0.836   0.928   0.881               CASEIN       Peptide sequence       EDVPSER   (SEQ ID NO: 14)   0.962   0.941   2.198   1.962               HQGLPQEVLNENLLR   (SEQ ID NO: 15)   0.828   0.701   1.736   2.090               FFVAPFPEVFGK   (SEQ ID NO: 16)   1.231   0.849   2.175   1.596               Average       1.007   0.830   2.036   1.883                  
 
         [0031]    Peptides whose sequences were confirmed by high-collision energy data are underlined in the above tables. Confirmation means that the probability of this peptide, given its accurate mass and the corresponding high-collision energy data, is larger than that of any other peptide in the database given the current fragmentation model. The remaining peptides are believed to be correct based on their retention time and mass compared to those for confirmed peptides. It was expected that there would be some experimental error in the results due to injection volume errors and other effects.  
         [0032]    When using BSA as an internal reference, the relative abundance of Glycogen Phosphorylase B in the first sample was determined to be 0.925 (first analysis) and 1.119 (second analysis) giving an average of 1.0. The relative abundance of Glycogen Phosphorylase B in the second sample was determined to be 1.244 (first analysis) and 1.292 (second analysis) giving an average of 1.3. These results compare favourably with the expected value of 1.  
         [0033]    Similarly, the relative abundance of Casein in the first sample was determined to be 0.980 (first analysis) and 1.111 (second analysis) giving an average of 1.0. The relative abundance of Casein in the second sample was determined to be 2.729 (first analysis) and 2.761 (second analysis) giving an average of 2.7. These results compare favourably with the expected values of 1 and 2-3.  
         [0034]    The following data relates to chromatograms and mass spectra obtained from the first and second samples. One peptide having the sequence HQGLPQEVLNENLLR (SEQ ID NO: 15) and derived from Casein elutes at almost exactly the same time as the peptide having the sequence LVNELTEFAK (SEQ ID NO: 4) derived from BSA. Although this is an unusual occurrence, it provided an opportunity to compare the abundance of Casein in the two different samples.  
         [0035]    FIGS.  11 A-D show four mass chromatograms, two relating to the first sample and two relating to the second sample. FIG. 11A shows a mass chromatogram relating to the first sample for ions having a mass to charge ratio of 880.4 which corresponds with the peptide ion (M+2H) ++  having the sequence HQGLPQEVLNENLLR (SEQ ID NO: 15) and which is derived from Casein. FIG. 11B shows a mass chromatogram relating to the second sample which corresponds with the same peptide ion having the sequence HQGLPQEVLNENLLR (SEQ ID NO: 15) which is derived from Casein.  
         [0036]    [0036]FIG. 11C shows a mass chromatogram relating to the first sample for ions having a mass to charge ratio of 582.3 which corresponds with the peptide ion (M+2H) ++  having the sequence LVNELTEFAK (SEQ ID NO: 4) and which is derived from BSA. FIG. 11D shows a mass chromatogram relating to the second sample which corresponds with the same peptide ion having the sequence LVNELTEFAK (SEQ ID NO: 4) and which is derived from BSA. The mass chromatograms show that the peptide ions having a mass to charge ratio of m/z 582.3 derived from BSA are present in both samples in roughly equal amounts whereas there is approximately a 100% difference in the intensity of peptide ion having a mass to charge ratio of 880.4 derived from Casein.  
         [0037]    [0037]FIG. 12A show a parent ion mass spectrum recorded after around 20 minutes from the first sample and FIG. 12B shows a parent ion mass spectrum recorded after around substantially the same time from the second sample. The mass spectra show that the ions having a mass to charge ratio of 582.3 (derived from BSA) are approximately the same intensity in both mass spectra whereas ions having a mass to charge ratio of 880.4 which relate to a peptide ion from Casein are approximately twice the intensity in the second sample compared with the first sample. This is consistent with expectations.  
         [0038]    [0038]FIG. 13 shows the parent ion mass spectrum shown in FIG. 12A in more detail. Peaks corresponding with BSA peptide ions having a mass to charge of 582.3 and peaks corresponding with the Casein peptide ions having a mass to charge ratio of 880.4 can be clearly seen. The insert shows the expanded part of the spectrum showing the isotope peaks of the peptide ion having a mass to charge ratio of 880.4. Similarly, FIG. 14 shows the parent ion mass spectrum shown in FIG. 12B in more detail. Again, peaks corresponding with BSA peptide ions having a mass to charge ratio of 582.3 and peaks corresponding with the Casein peptide ions having a mass to charge ratio of 880.4 can be clearly seen. The insert shows the expanded part of the spectrum showing the isotope peaks of the peptide ion having a mass to charge ratio of 880.4. It is apparent from FIGS. 12-14 and from comparing the inserts of FIGS. 13 and 14 that the abundance of the peptide ion derived from Casein which has a mass spectral peak of mass to charge ratio 880.4 is approximately twice the abundance in the second sample compared with the first sample.  
         [0039]    Kindly insert the following new section after the Detailed Description of the Preferred Embodiment.  
     
       
       
         1 
         
           
             16  
           
           
             1  
             10  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            1 

Phe Lys Asp Leu Gly Glu Glu His Phe Lys 
1               5                   10 

 
           
             2  
             11  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            2 

His Leu Val Asp Glu Pro Gln Asn Leu Ile Lys 
1               5                   10 

 
           
             3  
             15  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            3 

Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg 
1               5                   10                  15 

 
           
             4  
             10  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            4 

Leu Val Asn Glu Leu Thr Glu Phe Ala Lys 
1               5                   10 

 
           
             5  
             13  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            5 

Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val Arg 
1               5                   10 

 
           
             6  
             8  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            6 

Ala Glu Phe Val Glu Val Thr Lys 
1               5 

 
           
             7  
             7  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            7 

Val Leu Val Asp Leu Glu Arg 
1               5 

 
           
             8  
             9  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            8 

Thr Asn Phe Asp Ala Phe Pro Asp Lys 
1               5 

 
           
             9  
             9  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            9 

Glu Ile Trp Gly Val Glu Pro Ser Arg 
1               5 

 
           
             10  
             18  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            10 

Leu Ile Thr Ala Ile Gly Asp Val Val Asn His Asp Pro Val Val Gly 
1               5                   10                  15 

Asp Arg 

 
           
             11  
             11  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            11 

Val Leu Pro Asn Asp Asn Phe Phe Glu Gly Lys 
1               5                   10 

 
           
             12  
             14  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            12 

Gln Ile Ile Glu Gln Leu Ser Ser Gly Phe Phe Ser Pro Lys 
1               5                   10 

 
           
             13  
             11  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            13 

Val Ala Ala Ala Phe Pro Gly Asp Val Asp Arg 
1               5                   10 

 
           
             14  
             7  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            14 

Glu Asp Val Pro Ser Glu Arg 
1               5 

 
           
             15  
             15  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            15 

His Gln Gly Leu Pro Gln Glu Val Leu Asn Glu Asn Leu Leu Arg 
1               5                   10                  15 

 
           
             16  
             12  
             PRT  
             unknown  
             
               Chemically Synthesized  
             
           
            16 

Phe Phe Val Ala Pro Phe Pro Glu Val Phe Gly Lys 
1               5                   10