Beyond Quantitative Proteomics: Signal Enhancement of the a1 Ion as a Mass Tag for Peptide Sequencing Using Dimethyl Labeling Jue-Liang Hsu,† Sheng-Yu Huang,† Jen-Taie Shiea,‡ Wen-Ying Huang,‡ and Shu-Hui Chen*,† Department of Chemistry, National Cheng Kung University, No. 1 Ta-Hsueh Road, Tainan, 701, Taiwan, Department of Chemistry, National Sun Yat-Sen University, No. 70, Lienhai Road, 804 Kaohsiung, Taiwan. Received September 8, 2004
Stable isotope-based dimethyl labeling that produces a dimethyl labeled terminal amine or a monomethylated proline N-terminus by reductive methylation (Anal. Chem. 2003, 75, 6843-6852) was reported as a promising strategy for global quantitative proteomics because of the simplicity of the process and its fast and complete reaction. This labeling strategy provides a signal enhancement for the produced a1 ions, which are usually hard to detect among most of the nonderivatized fragments. To assist peptide sequencing, in this study, the enhanced a1 ion produced under either collision induced dissociation (CID) or post source decay (PSD) modes was further characterized and applied as a mass tag for fingerprinting the identity of N-terminal amino acid. On the basis of the analysis of standard peptides, tryptic digests of hemoglobin and cell lysates, it was proved that such signal enhancement occurred to a1 ions derived from all 20 of the amino acids residues and this phenomenon was explained based the formation of stable quaternary immoniun ions. Accurate determination of a1 ions was shown to increase the chance for peptide de novo sequencing and also provided higher confidence in the scores obtained when identifying a protein through database searching. In addition, the a1 ion was further demonstrated to be used as a universal tag for precursor ion scan in a Q-TOF instrument, leading to a greater number of peptide ions sequenced. Combined with the capability for differential quantitation, the stable isotope-based dimethyl labeling increases the usefulness of the labeling method for MS-based proteomics. Keywords: dimethyl stable isotope labeling • quantitative proteomics • de novo sequencing • mass spectrometry • reductive methylation
Introduction Mass spectrometry (MS)-based peptide sequencing is one of the most reliable techniques for the identification of proteins and their post-translation modifications. In this procedure, the peptide sequence is assigned from the analysis of the MS/MS fragments obtained from collision-induced dissociation (CID) or post-source decay (PSD) of the selected molecular ion. In most cases, however, full-length sequencing is not readily feasible for a diverse range of amino acid combinations in selected peptides because of incomplete or unpredictable fragmentations.1 The fragmentation pattern is affected by many factors; for example, the location of basic amino acid residues, such as Lys and Arg, is likely to direct the fragmentation pattern of a peptide ion, and the instrument setting parameters, such as the collision energy, may also affect the fragmentation.2 Most peptide sequences are derived by comparing a partial sequence obtained from experiment with sequences in protein databases that are derived mostly from the genome sequence3-5 but these databases are still either lacking or inadequate for some species. * To whom correspondence should be addressed. E-mail: shchen@ mail.ncku.edu.tw. † Department of Chemistry, National Cheng Kung University. ‡ Department of Chemistry, National Sun Yat-Sen University. 10.1021/pr049837+ CCC: $30.25
2005 American Chemical Society
In addition, post-translation modifications are not indicated in the genome database, which, therefore, may lead to mistakes in peptide sequencing or protein identification. Consequently, de novo peptide sequencing ; the complete interpretation of a peptide or protein sequence with only minimal assistance from genomic database ; remains one of the great challenges in proteomics research. For de novo peptide sequencing, some charged derivatives have been developed to simplify and to direct the fragmentation of peptides to facilitate the interpretation of the obtained MS/MS spectra.6-8 Chemical modifications on a peptide ion can enhance proton affinity, which, therefore, may increase the coverage of fragmentations in a certain peptide. For instance, Keough et al. used sulfonic acid derivatives, such as the watersoluble 3-sulfopropionic acid N-hydroxysuccinimide (NHS) ester, to react with peptide N-termini to form sulfonamides through a sulfonation process; the resulting sulfonic acidderivatized tryptic peptides were fragmented well under both electrospray ionization (ESI) and MALDI ionization conditions.9,10 The main drawback of this modification, however, is that the anionic sulfonic acid derivatized peptides exhibit a poor positive-ion sensitivity upon ionization: a reduction in sensitivity occurs by a factor of 10.1,10 Recently, Fales used divinyl sulfone (DVS) as a post-digestion modifier to enhance Journal of Proteome Research 2005, 4, 101-108
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research articles the intensity of the signal of the a1 ion produced in MS/MS and post-source decay.11 This enhanced signal can be used to decipher the first amino acid of a peptide; this information, which is normally unavailable in MS/MS spectra, is advantageous for de novo sequencing. In this reaction, DVS labels many amino acid residues including N-terminus, Lys, His, Cys, and this reaction may also produce isomeric products. The multiple labeling sites or isomeric products will, however, lead to complicated MS/MS spectra that may hinder the interpretation of peptide sequencing. In a previous study,12 stable isotopebased dimethyl labeling by reductive methylation (or reductive amination) was reported to be a promising strategy for global quantitative proteomics because of the simplicity of the process and its fast and complete (near 100% yield) reaction. In this modification, only the peptide N-terminus and the -amino groups of Lys residues are labeled by a pair of commercially available reagents, H2- and D2- formaldehyde to produce peaks that differ by 4 mass units for each derivatized isotopic pair. In addition to quantitation aspects, this labeling process also leads to signal enhancement for the a1 ions produced; these ions are usually hard to be detected among most of the nonderivatized fragments. Typically, the bn ions commonly observed in CID mass spectra13,14 have stable acylium structures that are formed by cyclization of protonated oxazolone molecules.15 Such a cyclization process, however, is not possible for the b1 ion and thus leading to a lack of b1 ions in CID spectra; this situation has prohibited direct sequencing of the N-terminal residue. In this study, the phenomena of enhanced a1 ions that arise from the labeling through reductive methylation are further characterized to explore its usefulness beyond quantitative proteomics.
Experimental Section Materials. Acetonitrile and formaldehyde (37% solution in H2O) were purchased from J. T. Baker (Phillipsburg, NJ). Trifluoroacetic acid (TFA), D,L-dithiothreitol (DTT), sodium cyanoborohydride, and hemoblobin, were provided by Sigma (St. Louis, MO). Iodoacetamide was purchased from Fluka (Buchs, Switzerland). R-Cyano-4-hydroxycinnamic acid (RCHCA), formaldehyde-d2 (20% solution in D2O) and the standard peptide (PLSRTLSVAAKK) were purchased from Aldrich (Milwaukee, WI). Formic acid (98∼100%) and sodium acetate were purchased from Riedel-de Hae¨n (Seelze). Ammonium hydroxide (ammonia solution, 29.8%) was purchased from TEDIA (Fairfield, Ohio, USA). Sequence grade-modified trypsin was purchased from Promega (Madison, WI). The water used in these experiments was obtained from an E-pure water purification system (Barnstead Thermolyne Co., Dubuque, IA). The E7 immortalized cell lysate obtained from Dr. N. H. Chou of the Pathology Department of the National Cheng Kung University was prepared as described previously.12 The tissue homogenate was prepared from Female Wistar rats (about 200∼350 g) on gestation day 18 (G18) and their uteri from antimesoemtrial regions (net weight about 13-15 mg) were removed. The preparation procedures were approved by the Animal Care and Use Committee of National Cheng Kung University (NCKU) in Taiwan. Formaldehyde is known to the state of California to cause cancer; special caution was taken including the use of surgical gloves and fume hood when handling formaldehyde. Tryptic Digestion of Standard Proteins. Proteins were reduced with DTT (10 mM, 2 µL) in 0.1M ammonium bicarbonate (pH 8.3) at 37 °C for 1 h and the resulting free cysteine 102
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residues were alkylated with iodoacetamide (10 mM, 4 µL) at 4 °C in the dark for 2 h. After digestion with trypsin (protein/ trypsin ) 50/1 in w/w) for overnight (∼14 h) at 37 °C, the resulting tryptic digest was further labeled using formaldehyde and sodium cyanoborohydride. Reductive Methylation of the Peptide and Tryptic Digests. The standard peptide (100 pmol) was diluted with 100 µL of sodium acetate buffer (100 mM, pH 5∼8). The solution was mixed with formaldehyde (4% in water, 4 µL), vortex, and then mixed immediately with freshly prepared sodium cyanoborohydride (600 mM, 4 µL). The mixture was vortex again and then allowed to react for 5 min. If necessary, ammonium hydroxide (4% in water, 4 µL) was added to consume the excess aldehyde. The hemoglobin digest (10 pmol/µL in 100 mM NaHCO3, 10 µL) was diluted with sodium acetate buffer (100 mM, pH 5∼7, 90 µL) and then it was labeled as described above. All the labeled peptides were monitored by MALDI instruments and were further analyzed by µLC-ESI-MS/MS. Mass Spectrometry. The MS data were obtained using a MALDI-TOF spectrometer equipped with a 337-nm N2 laser (M@LDI, Micromass, Manchester, UK). The PSD data were obtained using a MALDI-TOF instrument (AutoFlex II, Bruker Daltonics, Leipzig, Germany) equipped with a 337-nm N2 laser. The MALDI matrix was prepared by dissolving 4-cyanohydroxysuccinic acid (10 mg) in EtOH/MeCN (1:1, 1 mL) containing 0.1% TFA. The sample was acidified with 0.5 M HCl to minimize the salt effect16 and then it was mixed with the matrix at a ratio of matrix:HCl:sample ratio of 2:1:1 (v/v/v). The resulting mixture was deposited onto the target plate and dried before the detection. The dimethylated tryptic peptides were also analyzed using a Q-TOF micro spectrometer (Micromass, Manchester, UK) equipped with a nanoflow HPLC system (LC Packings, Amsterdam, Netherlands). Briefly, a tryptic digest solution (1 µL) was injected onto a column (NAN75-15-03-C18PM; 75 µm × 15 cm) packed with C18 beads (3 µm, 100 Å pore size, PepMap). Mobile-phase buffer A consisted of 0.1% formic acid in water; mobile-phase B consisted of 95% acetonitrile in 0.1% formic acid. The peptides were separated using a linear gradient of 0-70% solvent B over 40 min at a flow rate of 200 nL/min. Typically, 20-60 spectra were combined because the average peak duration for a peptide was about 20-60 s and each individual spectrum was acquired within 1 s at an interscan time of 0.1 s. For sequencing, the MS/MS spectra were obtained through a survey scan and the automated datadependent MS analysis was carried out using the dynamic exclusion feature built into the MS acquisition software. Peptide sequence assignment was performed using a peptide sequencing program (MassLynx, Micromass) either manually or processed through a database MS/MS ion search (Mascot MatrixScience) with variable reductive methylation of N-terminus and Lys. All results were further verified using manual interpretation. For precursor ion scans, two collision energies were used simultaneously (low collision energy ) 10 V; high collision energy ) 34 V) and the resulting CID spectra of selected precursors were acquired using two MS/MS channels. The molecular weight tolerance of the product ion was 10mDa and the threshold of intensity was 30 counts.
Result and Discussion Dimethyl Labeling. As indicated in eq 1, formaldehyde reacts with both the N-terminus and the -amino groups of Lys residues of a peptide to form Schiff bases, which are then reduced with sodium cyanoborohydride to form a secondary
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Figure 1. MALDI spectrum of a Pro-N-terminal peptide (PLSRTLSVAAKK). (A) before (m/z 1270.4), and (B) after labeling (m/z 1340.6). An asterisk denotes the labeling site.
amino groups that are relatively more reactive than their original primary amino groups. Subsequently, each of these more-reactive species reacts with another formaldehyde unit and is then reduced to form the end product bearing dimethylsubstituted tertiary amino groups.12
If the N-terminus is the secondary amino acid, proline, the reaction stops at the first step and forms a monomethylated amine, as expressed in eq 2.
These reactions can be proven by using a standard peptide (PLSRTLSVAAKK) that bears a Pro N-terminus and two Lys residues. As indicated in Figure 1A, the unmodified peptide ion (m/z 1270) is observed in the MALDI spectrum and is accompanied by broad bands that correspond to post-source decay products. After modification, as displayed in Figure 1B, the predominant ion (m/z 1340) corresponds to the peptide incorporating two Lys-dimethylation (28 Da × 2) sites and one N-Pro monomethylation (14 Da × 1) site. Except for its sodium adduct ions [M + Na]+, no detectable byproducts are observed in the spectra, which indicates that complete reductive methylation occurred. Fragmentation Pattern of Labeled Peptides. The fragmentation pattern of labeled peptides or tryptic digests was further investigated by LC-ESI-MS/MS and MALDI-PSD. Figure 2 depicts the fragment-ion spectrum of the labeled and unlabeled
tryptic peptides (VNNDEVGGEALGR) derived from hemoglobin, which have one labeling site: the valine residue at their N-termini. It is apparent that the a1 signal was greatly enhanced after modification (Figures 2A and B, asterisks denote the labeled site). Moreover, the intensity of a1 ion is usually the greatest and therefore, may be regarded as the base peak in the spectrum. For those peptides bearing Pro N-termini, the a1 ion is monomethylated rather than dimethylated, and so they undergo a mass increase of 14 rather than 28 Da. As indicated in Figure 3 for the Pro N-terminal peptide, *PLSRTLSVAA*K*K, the enhanced signal for the a1 ion (m/z 84.08) signal reflects the fact that the Pro residue was monomethylated. In addition to the enhancement of Val and Pro signals, the signal enhancement was confirmed to occur for a1 ions derived from all of twenty of the proteinogenic amino acids based on the analysis of standard peptides, tryptic digests of hemoglobin and E7 immortalized cell lysate. It was found that this phenomenon is universal for all of the kinds of peptides investigated, including those bearing missed cleaved lysine sites and multiple labeling sites. Similar results were also evidenced from post source decay performed in a MALDI instrument. As indicated in Figure 4, the PSD fragmentation pattern of the modified *LLVVYPWTQR ion (Figure 4B) is similar to that observed in the CID mode of an ESI instrument (Figure 4A). The a1 ion signal was enhanced substantially after the modification, which reveals that the fragmentation pattern associated with the N terminus-methylated peptides under the PSD mode is similar to that which occurs under the CID mode. In general, the above results indicate that this strategy can be applied to the analysis of wide range of peptides and also with different types of ionization source. It is also noticeable that in PSD spectrum the yn-1 signal is much lower than in CID. Actually, we have found that the yn-1 ions were enhanced occasionally even under the CID mode. Some ions were enhanced significantly but some were not. We are not sure about the cause of the occasional enhancement for yn-1 ions. Nonetheless, the sequencing information that yn-1 ions can provide is similar to what a1 ions can provide. Journal of Proteome Research • Vol. 4, No. 1, 2005 103
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Figure 2. CID spectra of (A) the native and (B) the labeled peptide (VNVDEGGEALGR derived from hemoglobin digest). The sequence of the first two amino acid residues of the native peptide cannot be assigned, but, on the other hand, the reductively methylated form can be sequenced completely.
Figure 3. CID spectrum of the reductive methylated Pro-N-terminal peptide (*PLSRTLSVAA*K*K).
The cause of the enhancement of a1 ion signal upon reductive methylation is not well understood but it may be explained by considering the following evidences. Typically, the bn ions that are commonly observed in CID mass spectra13,14 have stable acylium structures that are formed by cyclization of protonated oxazolone molecules.15 Such cyclization is not possible, however, for b1 ions, and thus, they are not generally 104
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observed in CID spectra. According to Harrison’s study, however, the b1 ion derived from unmodified methionine is a stable species because it forms a relatively stable cationic methyl R-amino-γ-thiobutyrolactone.17 When such ions, however, are dimethyl labeled, for example, *MFLSFPTT*K tryptic digest derived from hemoglobin, the spectra obtained display no detectable b1 ion, as indicated in Figure 5. This result implies
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Figure 4. Spectra of labeled peptide (*LLVVYPWTQR) derived from hemoglobin. These spectra were acquired under (A) CID and (B) PSD modes.
Figure 5. CID spectra of labeled peptides (*MFLSFPTT*K) derived from hemoglobin. Asterisks denote the labeling site.
that the R-aminoacylium ion, [RCH(NMe2)CO]+, of the dimethylated peptide exhibits a higher tendency to lose a CO molecule as described in Equation 3 than does the unmodified peptide.
Therefore, it is possible that any resulting b1 ions tend to form more-stable alkylated immonium a1 ions through the loss of carbon monoxide, which leads to substantial signal enhancement for labeled a1 ions after reductive methylation.
De Novo Sequencing. Theoretical masses of the a1 ions derived from the twenty amino acid residues after reductive methylation are summarized in Table S1 of the supplement material. The mass range of these a1 ions varies from 187.1235 (trytophan) to 58.0657 (glycine). Without modification, ion signals in this region are normally messy and almost no useful sequence information can be read. For the labeled peptides, however, the enhanced signals for the ions in this region, combined with accurate mass determination, can be used to fingerprint N-terminal residue. Except for the *L (leucine) and *I (isolucine) pair whose masses are indistinguishable under low energy CID conditions and the *R (arginine) and *K (lysine) pair, whose small mass difference (0.025 Da) is difficult to resolve when using a low-resolution instrument, the remaining sixteen a1 ions can be easily distinguished using well-calibrated mass spectrometer. For example, as indicated in Figure 2, the Journal of Proteome Research • Vol. 4, No. 1, 2005 105
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Figure 6. CID spectra of two tryptic peptides in E7 cell lysate. (A). *GVDEATIIDILT*K, a tryptic peptide derived from Annexin I; (B). *TFAPEEISAMVLT*K, a tryptic peptide derived from dnak-type molecular chaperone HSPA5 precursor.
assignment of the first two amino acid residues (VN or NV) of this tryptic peptide is difficult without the use of a database. After modification, however, the enhanced signal of the a1 ion provides an unambiguous assignment to be made for the first two amino acids: VN. Such de novo sequencing is particularly useful when analyzing unknown peptides in a complicated mixture. The full sequence assignment for an unknown peptide in the tryptic digest of E7 immortalised cell lysate can be deduced readily. As indicated in Figure 6A, except for the I and L residues whose masses are indistinguishable, the rest residues in this peptide sequence was unambiguously assigned de novo as *GVDEATIIDILT*K by using a manual peptide sequencing program (MassLynx, Micromass) and was further assigned to be Annexin I protein after performing a Mascot database search. In this case, the signal enhancement remained substantial even for the lowest molecular weight a1 ion, the N-terminal Gly residue (m/z 58.06). Similarly, another unknown peptide, derived from dnak-type molecular chaperone HSPA5 precursor, was readily assigned de novo to be *TFAPEEISAMVLT*K (Figure 6B). We have also included the MS/MS spectra of representative peptides deduced from another five proteinss elongation factor 2, human Adp-ribosylation factor 1, aspartate aminotransferase 2 precursor, 2-phosphopyruvate-hydratase alpha-enolase, and tyrosine 3/tryptophan 5-monoxygenase activation protein, in the supplement material (Figure S1-S5). The fragmentation pattern of these spectra allows a near complete de novo sequencing. Database-Assisted Sequencing. Peptide sequence assignment is most commonly performed through database MS/MS ion search (Mascot, MatrixScience) when a complicated biological system is analyzed. Normally, a score, which is affected by the quality of the acquired spectra, is assigned for each searched result to reflect the confidence of the accuracy of the match. Any enhancement of the signal of the a1 ion should 106
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improve the confidence of matching accuracy. To gain such an advantage, the scoring algorithm must take a ions into account in addition to b or y ions, but unfortunately, they are not included in the preset parameters for many instruments. On the basis of a Mascot search, we found that significant improvements in the acquired score occur when a ions were counted. For example, the peptide score for the ion, *VNNDEVGGEALGR derived from hemoglobin, increased from 52 to 71 when the contribution a ions was considered by the algorithm. Combined with accurate mass determinations, a novel searching algorithm may be proposed based on a1 ionspecific peptide database searching. First, the molecular weights of selected peptide ions are matched with those of tryptic peptides in the database under a suitable tolerance to find the many peptide candidates. Next, the accurate mass of the a1 ion is used to select among the N-terminal amino acids of all of the peptide candidates to narrow the number of candidates. Finally, the MS/MS spectra of the chosen candidates are matched with those in the database to deduce their sequences and any possible corresponding proteins. Such a searching algorithm will dramatically reduce the redundancy and increase the accuracy of database-assisted sequencing. Further development of this searching algorism is currently in progress. Precursor Ion Scan. Precursor ion scanning allows the efficient analysis of low-concentration samples by detecting specific fragments or neutral loss.18 The product ion used for the scan should be unique, however, so that it may assist in the data interpretation: for example, the phosphorylated immonium ion at m/z 216 from phosphotyrosine or the neutral loss of 98 Da (H3PO4) from phosphoserine and phosphothreonine residues.19,20 Moreover, the selected ion should also have appreciable intensity to allow an acceptable level of detection. The labeled a1 ions have enhanced intensity and characteristic masses that correspond to specific amino acids and, therefore,
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Figure 7. Base peak intensity (BPI) chromatograms of labeled hemoglobin tryptic digests (500 fmol) obtained using (A) low and (B) high collision energies. The denoted vales are the migration time corresponding to each peak; (C) MS/MS chromatogram of N-terminal Val containing peptides selected from precursor ion scan using the ion at m/z 100.11 ( 0.01 Da (a1 tag of Val). The m/z values corresponding to each peak are also indicated in below the value of their migration time and the sequences of N-termini Val containing peptides that were picked up during the scan mode are also indicated.
Figure 8. CID spectra of some Val-N terminal peptides selected from precursor ion scans (MS/MS spectra derived from Figure 7C) using the a1 tag ion at m/z 100.11 ( 0.01 Da.
they should meet these criteria. Hence, precursor ion scan was further applied for the analysis of selected N-terminal Val a1 ions (100.11 ( 0.01 Da) of a labeled hemoglobin digest. The results acquired from 500 fmol of labeled hemoglobin digest loaded on column are presented in Figures 7 and 8. Figure 7A,B
depicts the base peak intensity (BPI) chromatograms of the low (10 V) and high (34 V) collision-energy mass spectra, respectively. Figure 7C represents the MS/MS BPI chromatogram acquired using high collision energy, and it can be seen that the instrument has switched to this mode of operation several Journal of Proteome Research • Vol. 4, No. 1, 2005 107
research articles times during the experiment on the Val N-terminal a1 ion (100.11 ( 0.01 Da). A total of six Val N-terminal peptides were sequenced and four representative CID spectra are presented in Figure 8. It is worth noticing that only two Val N-terminal peptides were sequenced when the hemoglobin sample was analyzed using a regular LC-MS/MS survey scan at the same concentration and LC gradient, indicating an increase in the number of sequenced peptides when using precursor ion scan. We have further tested the helpfulness of N-termini a1 precursor ion scan for real samples using the tissue homogenate from late gestation rat uteri. Without any fractionation, seven peptides with the Val N-terminus from the labeled sample were identified by a1 precursor ion scan while there was only one Val N-terminal peptide from the unlabeled sample was picked up by the survey scan. We believe that the impact of the labeling and the use of a1 mass tag for precursor ion scan can be more noticeable when more separations are performed for sample preparation prior to MS analysis.
Conclusion As described in this study, the dimethyl labeling holds unique features beyond its use in quantitative proteomics, including providing higher confidence in the identification of proteins performed by either de novo sequencing or database-assisted searching and providing an universal a1 tag for mapping the N-terminal amino acid through precursor ion scan. Many other applications for MS-based proteomics may still be developed based on this labeling. One of the current developments is a study of N-terminal modifications that incorporate modified functional groups into the a1 tag. Whether or not these N-terminal modifications affect the reductive methylation process remains to be investigated. Finally, a novel search algorithm that incorporates the selection of a1 tag is also under development in order to reduce the redundancy and to increase the accuracy of search results.
Acknowledgment. This work was supported by the National Science Council under the National Program for Genomics Medicine (Grant NSC 93-3112-B-006-012-Y). We would also like to thank Prof. Nan-Haw Chow and Prof. MeiLing Tsai of the Medical College of National Cheng Kung University for preparing E7 cell lysate and the tissue homogenate from late gestation rat uteri.
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Supporting Information Available: MS/MS spectra of the dimethyl labeled peptide deduced from elongation factor 2, human Adp-ribosylation factor 1, aspartate aminotransferase 2 precursor, 2-phosphopyruvate-hydratase R-enolase, and tyrosine 3/tryptophan 5-monoxygenase activation protein in E7 cell lysate (Figures S1-S5). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Standing, H. G. Curr. Opin. Struct. Biol. 2003, 13, 595-601. (2) Dongre, A. R.; Jones, J. L.; Somogyi, A.; Wysocki, H. H. J. Am. Chem. Soc. 1996, 118, 8365-8374. (3) Mann, M.; Hendrickson, R. C.; Pandey A. Annu. Rev. Biochem. 2001, 70, 437-473. (4) Liebler, D. C. Introduction to Proteomics: Tools for the New Biology; Humana Press: Totowa, New Jersey; 2002. (5) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207. (6) Roth, K. D.; Huang, Z. H.; Sadagopan, N.; Watson, J. T. Mass Spectrom. Rev. 1998, 17, 255-274. (7) Sonsmann, G.; Romer, A.; Schomburg, D. J. Am. Soc. Mass Spectrom. 2002, 13, 47-58. (8) Keough, T.; Youngquist, R. S.; Lacey, M. P. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131-7136. (9) Keough, T.; Lacey, M. P.; Strife, R. J. Rapid Commun. Mass Spectrom. 2001, 15, 2227-2239. (10) Keough, T.; Youngquist, R. S.; Lacey, M. P. Anal. Chem. 2003, 75, 157A-165A. (11) Boja, E. S.; Sokoloski, E. A.; Fales, H. M. Anal. Chem. 2004, 76, 3958-3970.. (12) Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H. Anal. Chem. 2003, 75, 6843-6852. (13) Biemann, B.; Matsuno, T.; Caprioli, R. M.; Gross, M. L.; Seyama, T. Biological Mass Spectrometry. Present and Future; Wiley: New York, 1993. (14) Papayannopoulos, A. Mass Spectrom. Rev. 1995, 14, 49-73. (15) Yalcin, T.; Csizmadia, I. G.; Peterson, M. R.; Harrison, A. G. J. Am. Soc. Mass Spectrom. 1996, 7, 233-242. (16) Huang, S. H.; Hsu, J. L.; Morrice, N. A.; Wu, C. J.; Chen, S. H. Proteomics, 2004, 4, 1935-1938. (17) Tu, Y. P.; Harrison, A. G. Rapid Commun. Mass Spectrom. 1998, 12, 849-851. (18) Wilm, M.; Neubauer, G.; Mann, M. Anal. Chem. 1996, 68, 527533. (19) Bateman, R. H.; Carruthers, R.; Hoyes, J. B.; Jones, C.; Langridge, J. I.; Millar, A.; Vissers, J. P. C. J. Am. Soc. Mass Spectrom. 2002, 13, 792-803. (20) Salek, M.; Alonso, A.; Pipkorn, R.; Lehmann, W. D. Anal. Chem. 2003, 75, 2724-2729.
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