Anal. Chem. 2004, 76, 2748-2755
Guanidino Labeling Derivatization Strategy for Global Characterization of Peptide Mixtures by Liquid Chromatography Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Francesco L. Brancia,*,† Helen Montgomery,‡ Koichi Tanaka,‡ and Sumio Kumashiro†
Shimadzu Research Laboratory (Europe), Wharfside, Trafford Wharf Road, Manchester, M17 1GP, U.K., and Mass Spectrometry Research Laboratory, Shimadzu Corporation, Kyoto, Japan
Guanidination performed with isotopic isoforms of Omethylisourea was used in combination with reversedphase liquid chromatography (LC) matrix-assisted laser desorption/ionization to characterize, both qualitatively and quantitatively, protein mixtures. Synthesis of 13C- and 15N -labeled O-methylisourea sulfate produces a molecule 2 that is 3 Da heavier than the light isotopic variant. Protein mixtures containing identical components in different concentration are pooled together following parallel derivatization. Relative quantification of protein mixtures is achieved by mass spectrometry. A difference of 3 Da allows negligible interference between the two isotopic clusters for quantification of peptides up to 1400 Da. Under these conditions, the chromatographic resolution achieved allows separation of different pairs of derivatized peptides without altering the retention time of structurally identical isotopic isoforms. Concomitant isolation of both chemically modified precursors is followed by tandem mass analysis. Activation of the ions via collisions with an inert gas produces isotopically derivatized fragment ions, which appear as doublets in the product ion spectrum. Since the modification occurs on the C-terminal lysine, ions incorporating the guanidino moiety on the C-terminus can be distinguished from those containing the original unmodified peptide N-terminus. Knowledge of the location of the proton can be beneficial to data interpretation and peptide sequencing. The recent availability of several complete genome sequences and the development of “soft” ionization techniques in mass spectrometry has accelerated the process of large-scale identification of proteins.1,2 Although numerous sophisticated strategies have been introduced to investigate the nature and function of genes at the mRNA level (transcriptomics),3 proteome analysis * Corresponding author: E-mail:
[email protected]. Tel: 0044 161 888 4420. Fax: 0044 161 888 4421. † Shimadzu Research Laboratory (Europe). ‡ Shimadzu Corp. (Japan). (1) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269-95. (2) Chalmers, M. J.; Gaskell, S. J. Curr. Opin. Biotechnol. 2000, 11, 384-90. (3) Delneri, D.; Brancia, F. L.; Oliver, S. G. Curr. Opin. Biotechnol. 2001, 12, 87-91.
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presents more challenges due to the necessity to characterize systematically every protein with its own level of expression, modification, and interaction within multi-protein complexes. In proteome expression mapping,4 after protein identification is achieved by peptide mass fingerprinting and tandem mass analysis (MS/MS) in conjunction with collisionally activated decomposition, mass spectrometry can be exploited for quantifying alterations in protein abundance caused by internal or external perturbations of the cell. All mass spectrometry-based quantification methods rely on selectively labeling the sample with different stable isotopes.5 Isotopes can be introduced by metabolic labeling using labeled nutrients,6-10 by chemical derivatization of the peptide functional groups,11-13 or by enzymatic labeling through incorporation of 18O.14,15 In a typical experiment, each set of proteins/peptides is derivatized in parallel with isotopic isomers of the same chemical reagent/label. The samples are then pooled, and quantification is achieved by mass spectrometry following sample purification. These isotopic labeling strategies may be classified as internal standard methods in which a component from a control sample is used as standard against which the concentration of an unknown component is determined. A proteomics-oriented development in (4) Blackstock, W. P.; Weir, M. P. Trends Biotechnol. 1999, 17, 121-7. (5) Regnier, F. E.; Riggs, L.; Zhang, R.; Xiong, L.; Liu, P.; Chakraborty, A.; Seeley, E.; Sioma, C.; Thompson, R. A. J. Mass Spectrom. 2002, 37, 133-45. (6) Pasa Tolic, L.; Jensen, P. K.; Anderson, D. J.; Lipton, M. S.; Peden, K. K.; Martinovic, S.; Tolic, N.; Bruce, J. E.; Smith, R. D. J. Am. Chem. Soc. 1999, 121, 7949-50. (7) Pratt, J. M.; Robertson, D. H.; Gaskell, S. J.; Riba-Garcia, I.; Hubbard, S. J.; Sidhu, K.; Oliver, S. G.; Butler, P.; Hayes, A.; Petty, J.; Beynon, R. J. Proteomics. 2002, 2, 157-63. (8) Chen, X.; Smith, L. M.; Bradbury, E. M. Anal. Chem. 2000, 72, 1134-43. (9) Ong, S. E.; Kratchmarova, I.; Mann, M. J Proteome Res. 2003, 2, 173-81. (10) Krijgsveld, J.; Ketting, R. F.; Mahmoudi, T.; Johansen, J.; Artal-Sanz, M.; Verrijzer, C. P.; Plasterk, R. H.; Heck, A. J. Nat. Biotechnol. 2003, 21, 92731. (11) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-9. (12) Goodlett, D. R.; Keller, A.; Watts, J. D.; Newitt, R.; Yi, E. C.; Purvine, S.; Eng, J. K.; von Haller, P.; Aebersold, R.; Kolker, E. Rapid Commun. Mass Spectrom. 2001, 15, 1214-21. (13) Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047-57. (14) Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-42. (15) Reynolds, K. J.; Yao, X.; Fenselau, C. J. Proteome Res. 2002, 1, 27-33. 10.1021/ac030421+ CCC: $27.50
© 2004 American Chemical Society Published on Web 04/14/2004
external isotopic labeling methods was the invention of the isotopecoded affinity tags (ICAT).11 In both isotopic variants, the label is composed of three parts: first, an affinity tag (typically biotin), which is used to isolate ICAT-labeled peptides; second, a linker incorporating stable isotopes; and third, a reactive group with specificity toward thiol groups. Selective isolation of differentially labeled cysteine-containing peptides reduces the initial complexity of the peptide mixture. Recent improvements and variations of the ICAT method include the use of 13C for the heavy version16 and the replacement of the biotin moiety with photocleavable17 or acid-labile18 linker immobilized on a solid-phase support. Different labeling molecules were designed in order to target specific functional groups within the polypeptide chain. Novel derivatizing agents were synthesized for recognizing specifically the phosphate group,19 the indole of tryptophan,20 and primary amine.21 In the latter strategy, no methods possess the sufficient selectivity to specifically label only the N-terminus and avoid modification of lysine amino group.21-23 Of all the amino acid residues, lysine plays a crucial role in affecting the final appearance of a MS spectrum. In MALDI analysis, the lysine-containing peptides generated by tryptic digestion of proteins produce ions detected in lesser abundance than those incorporating arginine.24 Modification of the gas-phase basicity through selective conversion of the lysine into homoarginine (guanidination) has been showed to provide a beneficial effect on detection of peptide ions under MALDI analysis.25,26 In addition, the guanidination procedure has two advantages for peptide mass fingerprinting-based methods. First, it increases the overall number of fragments usable for database searching; second, comparing the derivatized ion signals with those present in the MS spectrum deriving from an underivatized protein mixture usually identifies the C-terminal amino acid of the tryptic digest, improving confidence in correct protein identification.27,28 Recently, a strategy for relative quantification of protein mixtures based on differential guanidination of C-terminal lysine residues of tryptic peptides followed by capillary liquid chromatography-electrospray tandem mass spectrometry was introduced.29 Although the method, termed mass-coded abundance tagging (MCAT), facilitates interpretation of product ion spectra of doubly charged ions by comparing the fragmentation pattern of treated and untreated analogues, determination of protein expression based on electro(16) Li, J.; Steen, H.; Gygi, S. P. Mol. Cell. Proteomics. 2003, 2 (11), 1198-1204. (17) Zhou, H.; Ranish, J. A.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2002, 20, 512-5. (18) Qiu, Y.; Sousa, E. A.; Hewick, R. M.; Wang, J. H. Anal. Chem. 2002, 74, 4969-79. (19) Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578-86. (20) Kuyama, H.; Watanabe, M.; Toda, C.; Ando, E.; Tanaka, K.; Nishimura, O. Rapid Commun. Mass Spectrom. 2003, 17, 1642-50. (21) Liu, P.; Reigner, F. E. J. Protein Res. 2002, 1, 443-50. (22) Che, F. Y.; Fricker, L. D. Anal. Chem. 2002, 74, 3190-8. (23) Beardsley, R. L.; Reilly, J. P. J. Protein Res. 2003, 1, 15-21. (24) Krause, E.; Wenschuh, H.; Jungblut, P. R. Anal. Chem. 1999, 71, 4160-5. (25) Brancia, F. L.; Oliver, S. G.; Gaskell, S. J. Rapid Commun. Mass Spectrom. 2000, 14, 2070-3. (26) Beardsley, R. L.; Karty, J. A.; Reilly, J. P. Rapid Commun. Mass Spectrom. 2000, 14, 2147-53. (27) Brancia, F. L.; Butt, A.; Beynon, R. J.; Hubbard, S. J.; Gaskell, S. J.; Oliver, S. G. Electrophoresis 2001, 22, 552-9. (28) Sidhu, K. S.; Sangvanich, P.; Brancia, F. L.; Sullivan, A. G.; Gaskell, S. J.; Wolkenhaue, O.; Oliver, S. G.; Hubbard, S. J. Proteomics 2001, 1, 136877. (29) Cagney, G.; Emili, A. Nat. Biotechnol. 2002, 20, 163-70.
spray response of the two samples suffers from the intrinsic differences in ionization efficiency existing between lysine- and homoarginine-containing peptides.30 Isotopic variants of labeling molecules forming a guanidino moiety with the lysine amino acid were prepared to circumvent discrepancies in response factor between the two derivatized peptides.31,32 The main limitation of these lysine-tagging methods arises from the complexity of the resulting MS spectrum caused by the higher distribution of lysine amino acids in proteins. In yeast, for instance, the abundance of lysine is ∼7% of the total number of amino acids in the proteome whereas cysteine and tryptophan account for less than 2%.7 In this paper, we present a guanidino-labeling derivatization strategy, termed GLaD, which combines differential guanidination with two isotopic variants and liquid chromatography followed by MALDI analysis (Scheme 1). The heavy version of O-methylisourea containing two 15N and one atom of 13C was synthesized ad hoc and used in conjunction with its light analogue for identification and subsequent quantification of protein mixtures. The initial complexity of the resultant sample was reduced through separation of the components by reversed-phase high-performance liquid chromatography (RP-HPLC), and since the type of isotopes used does not alter the retention time of the two analogues, both peptides are eluted together and subsequently collected in the same spot onto the MALDI target. From the relative intensity of their ion signals, it is possible to quantify by MS analysis the sample of interest. Concomitant activation of both ions in a quadrupole ion trap time-of-flight (QIT TOF) mass spectrometer generates product ion spectra in which y-type ions bearing the modification appear as doublets spaced by 3 Da. This allows differentiation between product ions containing the original peptide N-terminus and those retaining the charge on the modified C-terminal fragment of the peptide. Knowledge of the type of fragment ion observed in the MS/MS spectrum facilitates the correct determination of the peptide sequence. The GLaD strategy is a novel, effective, and economical method for protein identification and quantification and, by virtue of its simplicity, can be broadly used in comparative proteomics studies. EXPERIMENTAL SECTION Materials. Interleukin (VQGEESNDK) and horse apomyoglobin were purchased from Sigma (Poole, Dorset, U.K.), and a tryptic digest of glyceraldehyde-3-phosphate dehydrogenase was obtained from Michrom. Porcine trypsin was purchased from Promega (Southampton, Surrey, U.K.). Urea, dimethyl sulfate, sulfuric acid, and ammonium hydroxide were obtained from Aldrich (Milwaukee, WI). All solvents were HPLC grade and were purchased from Rathburn Chemicals (Walkerburn, Scotland, U.K.). Digestion of Proteins. The protein (1 nmol) was dissolved in 25 mM ammonium bicarbonate. Trypsin was added to give an enzyme-to-substrate ratio of 1:50 (w/w). The digest was kept at 37 °C overnight, after which digestion was terminated by addition of an equimolar amount of 1% formic acid. (30) Brancia, F. L.; Openshaw, M. E.; Kumashiro, S. Rapid Commun. Mass Spectrom. 2002, 16, 2255-9. (31) Peters, E. C.; Horn, D. M.; Tully, D. C.; Brock, A. Rapid Commun. Mass Spectrom. 2001, 15, 2387-92. (32) Mohammed, S.; Statea, I.; Brancia, F. L.; Oliver, S.; Gaskell, S. J. Proceedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics. Orlando, FL, June 2-6, 2002.
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Scheme 1. General Scheme of the GLaD Strategy
Formation of Light and 13C, 15N2-Labeled O-Methylisourea Sulfate. The method used was a modification of a procedure described by Mohammed et al.32 Urea (3.1 g, 0.048 mol) was added portionwise over 20 min to a stirred solution of dimethyl sulfate (4.44 mL, 0.048 mol) at 115 °C. After stirring for 1.5 h, the reaction was cooled to room temperature. Concentrated sulfuric acid (2.7 mL, 0.048 mol) was carefully dissolved in ether (25 mL). The resulting solution was then added to the oil of the reaction along with acetone. The mixture was cooled with vigorous stirring, and a solid precipitated was filtered and washed with acetone to yield the 13C, 15N2-labeled O-methylisourea sulfate hydrogen sulfate. An opaque solution of barium hydroxide monohydrate (2.05 g, 0.011 mol) was added to the aqueous solution of O-methylisourea. The colorless solution was then decanted and evaporated to dryness. The white solid obtained has a melting point of 157-158 °C (literature value, 159-160 °C): 1H NMR (250 MHz, DMSO-d6) δ 8.45 (br s, 4H, NH), 3.90 (d, J ) 4.2 Hz, 3H, OCH3); 13C NMR (62.9 MHz, DMSO-d6) δ 162.3 (t, J ) 23.3 Hzslitting caused by 2× 15N, 13C, 15NH), 57.2 (OCH3). Guanidination of Lysine Residues. The reaction was performed as described in previous literature25,33,34 with some modifications. Peptide solution (1-10 µM) was mixed with an aqueous solution of 0.5 M O-methylisourea adjusted to pH 11 with ammonium hydroxide. The resulting mixture was stirred overnight and the reaction stopped by the addition of an equal volume of 1% (v/v) aqueous TFA. A 2-µL aliquot of derivatized digest was desalted using ZipTip (Millipore, Watford, U.K.) following the manufacturer’s recommended protocol. Liquid Chromatography and MALDI Sample Collection. Reversed-phase capillary HPLC was performed on a LC-2010 (33) Bonetto, V.; Bergman, A. C.; Jornvall, H.; Sillard, R. Anal. Chem. 1997, 69, 1315-9. (34) Beardsley, R. L.; Reilly, J. P. Anal. Chem. 2002, 74, 1884-90.
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system (Shimadzu, Kyoto, Japan). Differentially labeled peptide solutions (1 µL) were prepared using 0.05% (v/v) aqueous TFA and loaded using a SIL-10AVP autosampler (Shimadzu). Sample preconcentration and desalting was accomplished using a Michrom Peptide CapTrap cartridge (0.5 mm (i.d.) × 2 mm), and components were separated on a Thermo Keystone Betabasic C18 column (0.32 mm (i.d.) × 10 mm). In gradient mode, the mobile-phase composition was 0.05% (v/v) TFA for the aqueous phase and 0.05 (v/v) %TFA and acetonitrile (9:1 (v/v)) for the organic phase. LC10ADVP HPLC pumps (Shimadzu) were used to deliver solvent at a flow rate of 200 µL/min. The composition of B was increased linearly for 50 min from 0 to 55%. Gradient profile was controlled by LCMS Solution 2.0 Software. The UV response of the column eluent was monitored at a wavelength of 220 nm. The system was configured to perform a postcolumn split. The sample eluted with a flow rate of 5 µL/min was delivered to an on-line AccuSpot LCMALDI spotting robot system (Shimadzu). Eluant was mixed with a saturated solution of R-cyano-4-hydroxycinnamic acid prepared in 50% (v/v) acetonitrile acidified with 0.1% (v/v) TFA. Ten spots of 1 µL each were deposited onto the MALDI target every minute. Mass Spectrometry. All MALDI -TOF mass spectra were acquired on an AXIMA-CFR plus instrument (Kratos Analytical, Manchester, England) equipped with a 1.2-m drift tube. Matrixassisted laser desorption/ionization of peptides was produced by pulses of UV light (λ ) 337 nm, 3-ns pulse width) generated by a nitrogen laser with a maximum pulse rate of 10 Hz. The ion source was typically held at 1 × 10-4 Pa. Spectra were collected in reflectron mode using a delayed extraction of 145 ns. The accelerating voltage was set to +20 kV. The reflectron voltage was set to +25 kV. The detector (microchannel plate) was connected with a 1-GHz (8-bit) transient recorder. Spectra were the sum of 100 profiles on the same sample spot. Each profile was the result of two consecutive single laser pulses. Acquisition
Figure 1. MALDI-TOF spectrum of a binary mixture containing 400 fmol of each labeled myoglobin digest. Guanidinated lysine terminal peptides are indicated with an asterisk. Homoarginine terminal peptides appear as doublets, and only the mass to charge value of the light version is displayed. In the inset, the isotopic variants of the modified ASEDLK*K* are separated by a shift of 6 Da due to the double derivatization of both lysine residues.
and data processing were controlled by Launchpad software version 2.3 (Kratos Analytical). The TOF was externally calibrated using an equimolar mixture of bradykinin, neurotensin, and human adrenocorticotropic hormone (ACTH 18-38). Data analysis was performed using raw data. Ion intensity was calculated from peak area for each isotopic envelope. MS/MS mass spectra were acquired on a AXIMA-QIT instrument (Kratos Analytical, Manchester, U.K.) previously described.35 Ions generated in the ion source are trapped and cooled using helium. The pressure in the trap is held at 4 × 10-3 Pa. The TOF mass analyzer is employed to acquire the data in both MS and MS/MS modes of operation. Prior to MS/MS analysis, precursor ions are isolated using the filtered noise field waveform applied to the end cap electrodes. The window used for precursor ion isolation was either 70 and 250 (resolution (m/∆m), where m corresponds to 1000). To induce fragmentation of the precursor ion, resonant excitation generated by a supplementary ac potential is applied to the end cap electrodes with a frequency matching the secular frequency of the ion. Following decomposition, the product ions are extracted into the TOF for mass analysis. In both MS and MS/MS modes, ions are pulsed into the TOF with an accelerating voltage of 10 kV. The detector is a microchannel plate and acquisition is made through 1-GHz transient recorder. The TOF mass analyzer was externally calibrated using fullerite (Aldrich) deposited directly onto the sample stage. RESULTS AND DISCUSSION General Overview of the GLaD Method. The proposed strategy relies on the use of two isotopic versions of the lysine(35) Martin, R. L.; Brancia, F. L. Rapid Commun. Mass Spectrom. 2003, 17, 1358-65.
labeling agent for quantifying proteins present in different amounts. It has been previously demonstrated that selectivity of guanidination is so high that unwanted derivatization of N-termini is not commonly observed.30;34 This advantageous feature precludes formation of byproducts, which can increase the complexity of the reaction mixture and diminish the sensitivity of the reaction. In this study, we utilized two versions of reagent to label lysine terminal peptide mixtures: O-methylisourea sulfate (light version) and its in-house synthesized 13C, 15N2-labeled analogue (heavy version). When the heavy version is employed, derivatization of the free lysine amino group results in the incorporation of two 15N atoms and one 13C within the guanidino moiety of the homoarginine. For higher mass peptides, a difference of 3 Da between the two adjacent derivatized peptide ions could generate overlapping between ions belonging to the two isotopic envelopes. To evaluate at what m/z value the overlapping between the two isotopic clusters becomes severe, negatively affecting the quantification, a tryptic digest of myoglobin was divided into two equal concentration aliquots and treated separately with both isotopic variants of O-methylisourea. Equal amounts of acidified reaction mixtures were pooled together and mixed with matrix. The resulting mixture containing 400 fmol of each labeled digest was deposited onto the target. Figure 1 illustrates the MALDI spectrum of the binary mixture. Analogue homoarginine terminal peptides appear as doublets separated by 3 Da. No traces of underivatized peptides are detected in the spectrum, and guanidination for both isotopic isoforms occurs as previously described.34 Tryptic peptides resulting from incomplete proteolytic digestion present derivatization of both lysine residues: for instance, in the modified peptide ASEDLK*K* at m/z 874.77, double derivatization is observed Analytical Chemistry, Vol. 76, No. 10, May 15, 2004
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Figure 2. Enlargement of the portions of mass spectrum, containing the differentially labeled peptides LFTGHPETLEK* (A) and HPGDFGADAQGAMTK* (B). For the peptide LFTGHPETLEK*, the ion species 12C59H93N16O18 and 12C5813CH9314N1415N2O18, at m/z 1313.6 and 1316.6, respectively, are the monoisotopic ions of the two peptides differentially guanidinated. For HPGDFGADAQGAMTK*, the monoisotopic ions of both guanidinated peptides (12C64H98N21O22S, m/z 1544.5, and 12C6313CH9814N1915N2O22S, m/z 1547.6) are also indicated in the spectrum.
inducing a shift of 84 and 90 Da, respectively (inset Figure 1). The portions of mass spectrum, which contain the differentially labeled peptides LFTGHPETLEK* and HPGDFGADAQGAMTK*, are illustrated in Figure 2. For the light version of the peptide ion LFTGHPETLEK* at m/z 1313.6, the fourth ion 12C5613C3H93N16O18 (2), of the first isotopic cluster overlaps the monoisotopic ion of the heavy analogue 12C5813CH9314N1415N2O18 (b). From the elemental composition, using the natural abundance of each isotope it is possible to calculate the relative abundance of 12C5613C3H93N16O18, so that its contribution to the ion detected at m/z 1316.6 can be determined. Its theoretical ion abundance corresponds to 5%. A similar calculation was performed for HPGDFGADAQGAMTK*, in which the light ion with three 13C isotopes (12C6113C3H98N21O22S) (1) overlaps the monoisotopic ion (12C6313CH9814N1915N2O22S) (9) of the heavy peptide ion at m/z 1547.5. The contribution of the light labeled ions on the ion species belonging to the isotopic envelope of the heavy labeled peptide is ∼8.5%. This observation suggests that, for peptides in the mass range 0-1400 Da, the use of reagents differing by 3 Da produces no significant contribution (1400 Da), in which overlapping of isotopic envelopes is detrimental to correct quantification, the contribution due to the heavier ions from the light isotopic cluster can be calculated a priori and subtracted from the heavier isotopic envelope. A proteomics strategy based on guanidination of lysine must cope with the inherent complexity arising from the high number of derivatized peptides generated. Specifically, when complex peptide mixtures are treated with O-methylisourea, several homoarginine terminal peptides are produced. To overcome difficulties in interpretation, LC-MALDI is an advantageous option for isolating differentially labeled components in fractions prior to sample preparation for MALDI analysis. The data presented have shown that, under the conditions used, isotopic isoforms of homoarginine terminal peptide
coelute. Since the heavy and light versions of a derivatized peptide cannot be resolved chromatographically, quantification does not suffer from factitious differences in concentration between the two differentially labeled peptides, caused by their different retention time. For MS/MS analysis, the production of several homoarginine terminal peptides is an advantage with respect to the other existing labeling techniques, which target less abundant amino acids.11,20 The possibility to obtain a larger number of homoarginine terminal peptides will provide a larger set of potential ion candidates available for tandem analysis. Under these circumstances, the larger number of MS/MS spectra obtainable from singly charged homoarginine terminal peptide ions can be utilized to achieve protein identification with higher confidence. Although the differential guanidination was not designed specifically for peptide sequencing of MALDI ions, isolation of both labeled precursors followed by activation can facilitate interpretation of product ion spectra of singly charged homoarginine terminal peptides. If tandem analysis is performed in MALDI instruments with the necessary requirements in term of mass resolution and mass measurement accuracy, the specific difference in mass can be detected in MS/MS spectra and all fragments containing the modified guanidino moiety can be distinguished from N-terminal fragment ions. In database searching, the incorporation of search parameters indicative of the type of fragment can improve search selectivity, providing a higher level of confidence in identification compared with that obtained with standard MS/MS searching strategies. However, the GLaD method is not restricted to the database searching approach for protein sequence determination, and the additional information provided could be exploited by de novo interpretation methods. Additionally, due to its intrinsic robustness, our procedure is amenable to implementation in conjunction with liquid chromatography electrospray tandem mass spectrometry and automation in sample-handling robot systems. In summary, this novel approach to proteome analysis is particular valuable in comparative studies in which the quantification of differentially expressed proteins is crucial for our understanding of the biological systems. ACKNOWLEDGMENT The authors are grateful to Shimadzu Corp. for funding this work. They also thank Dr. Simon Clayton and Dr. Andrew Regan for assistance in the synthesis of the heavy analogue of Omethylisourea. They acknowledge also Dr. Rachel Martin, Dr. Matt Openshaw, and Dr. Chris Sutton for the fruitful discussion during the preparation of the manuscript. Received for review December 22, 2003. Accepted March 3, 2004. AC030421+
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