Multiple Enzymatic Digestion for Enhanced Sequence Coverage of Proteins in Complex Proteomic Mixtures Using Capillary LC with Ion Trap MS/MS Gargi Choudhary,* Shiaw-Lin Wu, Paul Shieh, and William S. Hancock Thermo Finnigan, San Jose, California 95134 Received August 1, 2002
This study uses multiple enzyme digests to increase the sequence coverage of proteins identified by the shotgun sequencing approach to proteomic analysis. The enzymes used were trypsin, Lys-C, and Asp-N, which cleave at arginine and lysine residues, lysine, and aspartic acid residues, respectively. This approach was evaluated with the glycoprotein, tissue plasminogen activator, t-PA and gave enhanced sequence coverage, compared with a single enzymatic digest. The approach was then evaluated with a complex proteomic sample, namely plasma. It was found that trypsin and Lys-C were able to detect overlapping but distinct sets of proteins and a digital recombination of the data gave a significant increase in both the number of protein identifications as well as an increase in the number of peptides identified per protein (which improves the certainty of the assignment). Keywords: multiple enzymes • sequence coverage • tissue plasminogen activator • proteomics • plasma • ion-trap mass spectrometry
Introduction The analysis of complex proteomic samples is performed either by the separation of their protein mixtures by 2D-SDS/ PAGE, where the proteins are separated by MW and isoelectric point,1,2 or by enzymatic digestion of a set of proteins and analysis of the resulting peptide mixture. Multidimensional HPLC coupled to an ion-trap mass spectrometer is able to detect the resolved peptides by matching MS/MS spectra with predicted spectra from genomic or proteomic databases.3,4 This approach is particularly useful for very small samples where minimization of sample loss is key and the direct analysis of samples prepared by, for example, laser capture micro-dissection can enable the characterization of diagnostic proteins from transformed cells.5 In such analyses, however, proteins present at lower levels are detected (if at all) by the characterization of only one peptide and, thus, such an identification can be considered as only preliminary. Another problem with singlepeptide identification is that heterogeneity caused by processes such as alternative genomic splicing or posttranslational modification is unlikely to be identified. Also the use of peptide identifiers in sets of homologous proteins can be problematic and often results in significant additional interpretation efforts. Yates et al. have published the use of different enzyme digests for improved characterization of proteins due to the increased sequence coverage that can be obtained from the characterization of overlapping peptides.6 In their approach, three separate proteolytic digests are performed on isolated apohemoglobin variants. These digests are then combined into one sample tube and subsequently analyzed by a single LCMS/MS run. The increased complexity of characterization of * To whom correspondence should be addressed. Gargi Choudhary, Ph.D., Thermo Finnigan, 355 River Oaks Parkway, San Jose, CA 95134. 10.1021/pr025557n CCC: $25.00
2003 American Chemical Society
combined digests in a single analysis parallels some of the complexity of individual proteomic analyses. Complete sequence coverage of the proteins was achieved by optimizing conditions of sample preparation, i.e,, digestion times and buffer conditions for given proteases, chromatography and automated mass spectrometric data collection. In the present study, we have explored the approach of multiple enzymatic digests for the characterization of a recombinant DNA derived (rDNA) protein, tissue plasminogen activator (t-PA). A single enzyme will not yield full coverage, for example, in the presence of clevage sites too close to the N or C terminus. The peptide digests obtained from different proteases were run in parallel, contrary to Yates approach, and the data was combined post acquisiton to obtain enhanced sequence coverage. This approach was then applied to characterization of plasma proteins with the view of achieving improved identification of proteins. As part of the study, advantages of performing separate digests with the digital recombination of resulting files is compared with manual combination of the separate digest samples and performing the analysis in a single HPLC run.
Materials and Methods Materials. DL-dithiothreitol and iodoacetic acid were obtained from Pierce (Rockford, IL). HPLC grade water was obtained from J. T. Baker (Phillisburg, NJ) and UV grade acetonitrile was obtained from Burdick and Jackson (Muskegon, MI). “Suprapur” formic acid (98-100%) was obtained from EM Science (Gibbstown, NJ). Guanidine hydrochloride, ammonium bicarbonate, ethylenediamine tetraacetic acid (EDTA disodium 0.5M), and tris-HCl were obtained from Sigma Chemical Company (St. Louis, MO). Sequencing-grade modified trypsin was obtained in 100 µg aliquots from Promega Corporation (Madison, WI). Sequencing grade endoproteinase Lys-C and Journal of Proteome Research 2003, 2, 59-67
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Figure 1. Base peak chromatogram illustrating peptide map of recombinant tissue plasminogen activator obtained by micro LC-MS/ MS analysis of trypsin digest. In the tryptic map, the MS/MS scan at 33.2 min was matched against human database and assigned to peptide VEYCWCNSGR. The MS/MS fragmentation pattern of this peptide with labeling of the major fragmentation sites is also shown.
Asp-N were obtained from Wako Pure Chemical Industries Ltd. (Richmond, Virgina). Plasma was obtained from Sigma Chemical Company (St. Louis, MO). Enzymatic Digestion. Lyophilized rt-PA (1 mg) was reconstituted in 1 mL reduction buffer (6 M guanidine hydrochloride, 100 mM ammonium bicarbonate, pH 8.5) and mixed with 10 µl of 1 M dithiothreitol (DTT). The mixture was incubated at 37 °C for 30 min and then 25 µl of iodoacetic acid (1 M in 1 M NaOH solution) was added and incubated for an additional 30 min at room temperature in the dark. Finally, 100 µl of 1 M DTT was added to quench the reaction yielding reduced and carboxymethylated rt-PA (RCM rtPA). Similar protocol was adopted for obtaining reduced and carboxymethylated plasma (RCM plasma, 5 mg/mL). Trypsin Digestion. The RCM rt-PA/plasma was exchanged into 100 mM ammonium bicarbonate buffer, pH 8.5. The buffer exchanged RCM rtPA (1 mg)/plasma (5 mg) sample was incubated with trypsin (40:1, substrate to enzyme) and incubated at room temperature. A second dose of trypsin was added after 8 h and the reaction continued for a total of 24 h. Lys-C Digestion. The RCM rt-PA/plasma was exchanged into 25 mM Tris-HCl, pH 8.5, and 1 mM EDTA. The buffer exchanged RCMrtPA (1 mg)/plasma (5 mg) was mixed with Lys-C (40:1, substrate to enzyme) and incubated at 37 °C overnight. Asp-N Digestion. The RCM rt-PA was exchanged into 50 mM phosphate buffer, pH 8.0. The buffer exchanged rt-PA was mixed with 100 µg of Asp-N and incubated at 37 °C overnight. Chromatography. Chromatography was performed using a 60
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Surveyor LC system (Thermo Finnigan, San Jose, CA) on a 150 µm × 10 cm column packed with 5 µm, Zorbax C18 stationary phase. The pump flow rate is split 1:100 for a column flow rate of 1.5 µl/min. The column effluent is directly electrosprayed using the orthogonal metal needle source without further splitting. Mobile phase A is 0.1% formic acid in water, and the B mobile phase is 0.1% formic acid in acetonitrile. The separation of peptides obtained by enzymatic digest of rt-PA was achieved with a gradient of 2-60% B over 90 min. A gradient from 2-60% B in 240 min was used for separation of enzymatic digests of plasma. Mass Spectrometry. The column effluent from the reversed phase column was analyzed by LCQ Deca XP ion-trap mass spectrometer. The micro-electrospray interface uses a 30 µm metal needle that is orthogonal to the inlet of the LCQ. The temperature of the transfer capillary is set at 120 °C, whereas the spray voltage at the metal needle is set at 2.6 kV, and the sheath gas is optimized at 10 units. For human plasma analysis, the mass spectrometer was set such that one full MS scan was followed by three MS/MS scans on the three most intense ions from the MS spectrum. In addition, peptide ions for which sequencing information had been collected were dynamically excluded from reanalysis for 5 min. Protein Identification. The acquired MS/MS spectra were automatically searched against protein database for human proteins using the TurboSEQUEST software. The SEQUEST search results were initially assessed by examination of the Xcorr (cross correlation) and ∆Cn (delta normalized correlation) scores. The Xcorr function measures the similarity between the
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unified ranking score. This algorithim has been incorporated into Bioworks 3.09 which is a new version of TurboSEQUEST.
Results and Discussion
Figure 2. Illustration of sequence coverage by capillary LC-MS/ MS analysis of trypsin digest of room temperature-PA. (Amino acids marked in red are identified, blue are part of a glycopeptide and black are not identified).
mass-to-charge ratios (m/z) for the fragment ions predicted from amino acid sequences obtained from the database, and the fragment ions observed in the MS/MS spectrum. The ∆Cn score is obtained by normalizing the Xcorr values to 1.0 and observing the difference between the first- and second-ranked amino acid sequences.7 Thus, the ∆Cn score discriminates between high quality and noisy spectra although both may match a theoretical spectra. As a general rule, an Xcorr value of greater than 2.0 for a doubly charged ion (>1.5 for singly charged ion) and ∆Cn greater than 0.1, was accepted for positive identification.8 TurboSEQUEST output results were summarized using newly developed algorithm where the three matching factors (Sp, Xcorr, and δCn) are used to construct a
Enhanced Sequence Coverage for a Recombinant Protein. Recombinant tissue plasminogen activator is a 527 residue glycoprotein that upon trypsin digestion theoretically yields 51 peptides.10 Contained in these 51 peptides are three N-linked glycopeptides present at asparagine 117 (peptide T11), 184 (peptide T17), and 448 (peptide T45). Peptide T11 has been shown to have attachments of high mannose structures, whereas glycosyation sites of peptides T17 and T45 have attachments of complex type of carbohydrates.11 Peptide T17 is glycosylated in 50% of the rt-PA molecules and should be present in the tryptic map in both glycosylated and unglycosylated forms.12 Figure 1 shows base peak chromatogram illustrating the peptides obtained from data dependent LC-MS/MS analysis of tryptic digest of rt-PA. The map contains mass and fragmentation pattern information on the eluting peptides. Figure 1 also shows the MS/MS fragmentation pattern, with labeling of the major fragmentation sites, of the peptide eluting at 33.2 min. Upon a TurboSEQUEST search of this scan against human database a sequence of VEYCWCNSGR was assigned to this peptide. Figure 2 shows that the protein was identified as t-PA when the entire product ion spectra from the LC-MS/MS analysis of tryptic digest mixture were used by TurboSEQUEST software to search the human protein database. The peptides
Figure 3. Base peak chromatogram illustrating peptide map of recombinant tissue plasminogen activator obtained by micro LC-MS/ MS analysis of Lys-C digest. In the peptide map, the MS/MS scan at 42.3 min was matched against human database and assigned to peptide PWCYVFK. The MS/MS fragmentation pattern of this peptide with labeling of the major fragmentation sites is also shown. Journal of Proteome Research • Vol. 2, No. 1, 2003 61
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Figure 4. Base peak chromatogram illustrating peptide map of recombinant tissue plasminogen activator obtained by micro LC-MS/ MS analysis of Asp-N digest. In the peptide map, the MS/MS scan at 43.5 min was matched against human database and assigned to peptide DIALLQLKS. The MS/MS fragmentation pattern of this peptide with labeling of the major fragmentation sites is also shown.
highlighted in red were immediately identified using TurboSEQUEST representing 65% coverage of the protein. The peptides labeled in blue are the glycosylated peptides whereas the peptides in black are not observed. It can also be seen from Figure 2 that the peptides, SYQVICRDEK, at the N terminal end and, DNMRP, at the C terminal end are not fully identified by trypsin digestion. This makes the task of detecting N/C-terminal processing difficult. Additionally, peptides NPDR, AGK, NR, R, SPGER, IK, HR, R, SPGER, TYR, and SDSSR are not identified because they either are not retained on the reversed phase column or fall below the lower mass limit set for the mass spectrometer. Glycopeptides T11 and T45 are not identified because their mass is greater than the predicted value whereas glycopeptide T17 (YSSEFCSTPACSEGNSDCYFGNGSAYR) is easily identified since it also exists in the nonglycosylated form. Figure 3 shows the base peak chromatogram illustrating the peptides obtained from data dependent LC-MS/MS analysis of Lys-C digest (cleaves predominately at lysine residues) of rt-PA. MS/MS fragmentation pattern of the peptide eluting at 42.3 min and identified as PWCYVFK based on a search against human database is also illustrated. A combination of TurboSEQUEST search as well as biomass deconvolution gives a sequence coverage of 62.8% for rt-PA using Lys-C as the enzyme. Figure 4 shows base peak chromatogram illustrating 62
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the peptides obtained by Asp-N (cleaves N-terminally at aspartic acid residues) digest of rt-PA. The inset in the figure shows MS/MS fragmentation pattern of the peptide eluting at 43.5 min and identified as DIALLQLKS. A combination of TurboSEQUEST search and biomass deconvolution for Asp-N enzyme shows sequence coverage of 34.9%. It is obvious that none of the enzymatic digests alone gives complete sequence coverage for the recombinant protein under consideration. A combination of the trypsin and Lys-C digests shows combined sequence coverage of 88.2% (data not shown). There is also 62.8% redundancy in the coverage as a result of running two instead of one digest of the recombinant protein. This redundancy provides further confirmation of the correct sequence which is important in determining possible sites and types of variations resulting from single nucleotide polymorphisms or posttranslational modifications. A combination of the peptides identified by the trypsin, Lys-C and Asp-N enzymatic digestion results in enhanced sequence coverage of 93.9% for the rt-PA and is illustrated in Figure 5. Analysis of Figure 5 shows that both the N-terminal peptide (SYQVICRDEK) and the -Cterminal peptide (VTNYLDWIRDNMRP) are now identified. Also, Asp-N provides coverage of protein sequence in the regions that are not covered by either trypsin or Lys-C, i.e., amino acids 248-267. Only the peptides marked in red are not
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Figure 5. Enhanced sequence coverage of recombinant tissue plasminogen activator obtained by micro LC-MS/MS analysis of three (trypsin, Lys-C, Asp-N) parallel enzymatic digests.
Figure 6. Base peak chromatogram illustrating peptide map of human plasma obtained by micro LC-MS/MS analysis of tryptic digest. In the tryptic map, the MS/MS scan at 46.2 min was matched against human database and assigned to peptide EGYYGYTGAFR. The MS/MS fragmentation pattern of this peptide with labeling of the major fragmentation sites is also shown.
identified since these are the glycosylated peptides. Identification of glycosylated peptides is a challenging task due to the heterogeneity of glycoforms. For quality control purposes, because the structure and hence the mass of the main glycoform of the peptide is known, the extracted ion chromatogram can be used to extract the ion of interest.12 MSn capabilities of the ion trap mass spectrometer can also be used for determination of detailed structure of the attached glycopeptide for the protein under consideration.13 Enhanced Sequence Coverage for Plasma. Although the multienzyme approach can be valuable for rapid characterization of a protein pharmaceutical, the next step in this study
was to apply this approach to a complex proteome sample, such as human plasma. In a previous publication, we have shown that the characterization of proteins in such a matrix is facilitated by the use of 2-D chromatography (ion exchange and reversed phase HPLC) over a 1-D reversed phase chromatography.14 The improved chromatographic resolution obtained from 2D chromatography allows the mass spectrometer to perform more MS/MS identifications per chromatographic peak. In this study, we have used the 1-D reversed phase separation and investigated the use of a protease with different cleavage specificity to generate novel peptides that could offer Journal of Proteome Research • Vol. 2, No. 1, 2003 63
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Figure 7. Base peak chromatogram illustrating peptide map of human plasma obtained by micro LC-MS/MS analysis of Lys-C digest. In the peptide map, the MS/MS scan at 64.3 min was matched against human database and assigned to peptide EGYYGYTGAFRCLVEK. The MS/MS fragmentation pattern of this peptide with labeling of the major fragmentation sites is also shown.
additional characterization opportunities. The current approach of using multiple proteases to characterize complex proteomic mixtures is infact complimentary to the use of multidimensional chromatography. Samples of complex proteomic mixtures digested by multiple enzymes can be analyzed by either 1-D or 2-D chromatography with the aim of obtaining enhanced sequence coverage. In the present study, we used 1-D chromatography for the sake of simplicity. Figures 6 and 7 show the complex elution profiles obtained by LC-MS/MS analysis of trypsin and Lys-C digests of plasma, respectively. The analysis was performed with 75 µg of digested plasma loaded on a 150 µm × 100 mm column packed with a C18 stationary phase. The gradient was from 2 to 60% B in 240 min. As described by Yates et al.,6 features of data dependent acquisition, nth most intense ion, dynamic exclusion and isotope exclusion are incorporated to increase the efficiency of data collection. In our MS acquisition method, the data is acquired using four scan events. The first scan event acquires an MS scan followed by three scan events for MS/MS on the top three ions. Dynamic exclusion with repeat count, repeat duration, exclusion duration and exclusion mass width of 1, 0.5 min, 5 min, and 3 u respectively were set to prevent multiple acquisitions of MS/MS spectra for a given m/z value. Table 1 shows a list of top 50 proteins obtained by capillary LC-MS/MS analysis of trypsin digest of plasma. In the tryptic map of plasma, MS/ 64
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MS scan at 46.2 min was matched against human database and assigned to peptide EGYYGYTGAFR (inset in Figure 6), whereas the scan at 64.3 min in the map of Lys-C digest was assigned to peptide EGYYGYTGAFRCLVEK (inset in Figure 7). Because Lys-C cleaves at only the lysine residue a larger fragment of the peptide was identified in its digest of plasma. Additionally, upon analysis of entire product ion spectra of the trypsin and the Lys-C maps by TurboSEQUEST, both these peptides were assigned to protein transferrin, independently. In these analyses, trypsin identifies a greater number of proteins than Lys-C (193 vs 149 proteins, respectively), which is probably related to the greater number of peptides produced by the more frequent cutter. Typsin and Lys-C digests of human plasma were subsequently mixed (1:1) and 75 µg of this sample was analyzed by LC-MS/MS. A total of 263 proteins were analyzed with a gradient running from 2 to 60% B in 360 min. Because the high level proteins such as albumin, globulins, lipoproteins, and transferrin have not been removed before digestion, one could expect that most if not all low level peptides would be obscured by the more abundant peptides. Despite this complexity, there are regions in the elution profile which are relatively open and in a previous study15 one such region was exploited to characterize growth hormone, which is present in nanomolar concentrations in plasma, via the N-terminal tryptic peptide. Thus, one could expect that trypsin and LysC would
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Figure 8. Graph illustrating the number of unique peptides from each individual protein identified upon analysis of trypsin and Lys-C digests of human plasma. (20 common proteins are illustrated in this graph).
Figure 9. Graph illustrating the number of unique peptides from each individual protein identified upon analysis of trypsin and Lys-C digests of human plasma. (Common proteins with less than seven peptides are illustrated in this graph).
detect overlapping sets of proteins in a complex proteome analysis. Figure 8 shows that this is indeed the case for 20
common proteins in the analysis of plasma. The figure illustrates the number of unique peptides identified for an Journal of Proteome Research • Vol. 2, No. 1, 2003 65
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Table 1. List of Top 50 Proteins Obtained by Capillary LC-MS/ MS Analysis of Tryptic Digest of Human Plasma
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
protein description
unique peptides
serum albumin precursor transferrin precursor fibrinogen, β-chain preproprotein R-2-macroglobulin precursor unnamed protein product apolipoprotein A-I precursor complement C3 presursor fibrinogen, γ- chain Ferroxidase precursor A chain A, crystal structure of soluble human Igg1 Fc fragment R-1-acid-glycoprotein 1 precursor fibrinogen, R-chain immunoglobulin heavy chain unnamed protein product R-2-HS-glycoprotein precursor β-2-glycoprotein I precursor IG-R-1 chain C region histidine-rich glycoprotein precursor haptoglobin-related protein [Homo sapiens] hemopexin [Homo sapiens] group-specific component (vitamin D binding protein) complement component 4B L chain L, antithrombin Ig κ-chain C region immunoglobulin heavy chain constant region vitronectin complement component 1 inhibitor unnamed protein product A chain A, tissue factor pathway inhibitor A chain A, structure of human transthyretin complement component 4-binding protein, R complement factor H precursor AF151072_1 HSPC238 [Homo sapiens] haptoglobin [Homo sapiens] R-1-antitrypsin precursor fibrinogen RA AF311103_1 TRPM-2 [Homo sapiens] anti-ds DNA immunoglobulin λ-chain variable region Ig λ-chain (Ke-O+) immunoglobulin heavy chain constant region γ-4 Ig G1 H Nie R-1B-glycoprotein immunoglobulin heavy chain immunoglobulin κ-chain variable region R-1-acid glycoprotein 2 apolipoprotein C-III precursor β-globin AF304164_1 keratin 1 [Homo sapiens] choriogonadotropin β-chain precursor complement component 3 precursor
39 26 14 17 12 9 12 11 6 6 5 9 5 2 2 5 7 8 8 6 6 4 4 1 2 3 3 2 2 2 2 4 3 2 1 1 3 2 3 2 1 2 1 1 4 1 2 1 1 1
individual protein upon digestion of human plasma by trypsin (blue bars) and Lys-C (pink bars). For example, 39 peptides are identified for human serum albumin upon analysis of trypsin digest of plasma, whereas 27 peptides are identified upon analysis of Lys-C digest. A digital combination of the results from trypsin and Lys-C (indicated by yellow bars in Figure 8) increases the number of unique peptides identified for human serum albumin to 54, thereby increasing sequence coverage for this protein. This is in contrast to the results obtained from analysis of the mixture of the two digests. Only 42 unique peptides were identified for human serum albumin by analysis of the mixture in comparison to the 54 obtained by analysis of the digests in parallel. Figure 8 shows an 66
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enhancement in sequence coverage for 16 of the 20 proteins listed. The improved coverage is important for not only improved probability of identification, but also increases the likelihood of the detection of structural variants generated by processes such as alternative splicing and posttranslational modifications. The characterization of certain proteins, however, is often limited to the detection of only a few peptides and again the use of multiple enzyme digests increases the number of peptides that are identified. Figure 9 shows a graph of number of unique peptides identified for a set of 19 proteins with only a few peptides (less than 7) identified per protein. For some of these proteins only one peptide is identified by LC-MS/MS analysis of either Lys-C (pink bar) or trypsin digest (blue bar) of human plasma. A digital recombination of the data (yellow bar) obtained from the two digests again shows an increase in the number of peptides identified for 15 of the 19 proteins shown in Figure 9. An example is complement component 3 protein. VLLDGVQNPR is identified as a doubly charged peptide for this protein (DeltaCn 0.47, Xcorr 3.4) upon analysis of trypsin digest of plasma, whereas upon analysis of Lys-C digest VLLDGVQNPRAEDLVGK is identified as a doubly charged peptide (DeltaCn 0.49, Xcorr 3.5) for complement component 3. Thus, a combination of trypsin and Lys-C digests increases the number of peptides identified for complement component 3. Also, interestingly the peptide identified from Lys-C digest has an overlapping sequence with that identified by trypsin. Thus, the confidence in the positive identification of complement component 3 as a one of the plasma proteins is further increased. For 4 of the 19 proteins listed in Figure 9 there is no increase in the number of the peptides identified. However, simultaneous determination of the same peptide in both digests increases the confidence in identification for the protein. Thus, in these studies the digital recombination of data from different enzymatic digests gave an increased confidence in identification of peptides than the alternative of combining the digests before a single LC/MS run. Such a result can be attributed to the increase in identifications achieved by the mass spectrometer with a reduced mixture complexity at a given time point in the case of the digests analyzed separately.
Acknowledgment. The authors are grateful for the following collaborations: software development in Request (Dr. Pavel Bondarenko), the administrative support of Ms. Debbie Krantz, and the guidance of Dr. Ian Jardine. The authors also recognize the assistance of other colleagues, and Dr. Iain Mylchreest and other members of product development team. References (1) O’Farrell, P. H. J. Biol. Chem. 1975, 250, 4007-4021. (2) O’Farrell, P. Z.; Goodman, H. M.; O’Farrell, P. H. Cell 1977, 12, 1133-1142. (3) Hunt, D. F.; Yates, J. R., III; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. 1986, 83, 6233-8238. (4) Yates, J. R., III; McCormack, A. L.; Eng, J. Anal. Chem. 1996, 68, 534A-540A. (5) Wu, S.-L.; Hancock, W. S.; Goodrich, G. G.; Kunitake, S. T. An approach to the proteomic analysis of a breast cancer cell line (SKBR-3); 2002, Manuscript submitted for publication. (6) Gatlin, C. L.; Eng, J. K.; Cross, S. T.; Detter, J. C.; Yates, J. R., III Anal. Chem. 2000, 72, 757-763. (7) Eng, J. K.; McCormick, A. L.; Yates, J. R., III J. Am. Mass Spectrom. 1994, 5, 976-989. (8) Ducret, A.; Van Oostveen, I.; Eng, J. K.; Yates, J. R., III; Abersold, R. Protein Sci. 1998, 7, 706-719. (9) ThermoFinnigan Product Support Bulletin, B-1033, Feb 2002, “ Xcalibur Bioworks 3.0”.
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with Ion Trap MSn, ThermoFinnigan application report no. 300, January, 2002. (14) Hancock, W. S.; Choudhary, G.; Wu, S.-L.; Shieh, P. Am. Laboratory 2000, 32, 20-22. (15) Wu, S.-L.; Amato, H.; Biringer, R.; Choudhary, G.; Shieh, P.; Hancock, W. S. Targeted proteomics of low level proeins in human plasma by LC/MSn: using human growth hormone as a model system, 2002, J. Proteome Res. 2002, 1, 5, 459-465.
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