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Derivatization with 1-Pyrenyldiazomethane Enhances Ionization of Glycopeptides but Not Peptides in Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Junko Amano,*,† Takashi Nishikaze,† Fumio Tougasaki,† Hiroshi Jinmei,† Ichiro Sugimoto,† Shu-ichi Sugawara,‡ Masaya Fujita,‡ Kenji Osumi,‡ and Mamoru Mizuno‡ Laboratory of Glycobiology, and Laboratory of Glyco-Organic Chemistry, The Noguchi Institute, 1-8-1 Kaga, Itabashi, Tokyo, 173-0003, Japan Glycoproteomics holds the promise of new advances in medical technology. However, mass spectrometry has limitations for the structural determination of glycosylated peptides because the hydrophilic nature of the oligosaccharide moiety in glycopeptides is disadvantageous for ionization, and glycopeptides ionize much less readily than nonglycosylated peptides. Therefore, conventional proteomics tools cannot detect altered glycosylation on proteins. Here, we describe an on-plate pyrene derivatization method using 1-pyrenyldiazomethane for highly sensitive matrix-assisted laser/desorption ionizationtandem mass spectrometry (MALDI-MSn) of glycopeptides in amounts of less than 100 fmol. This derivatization is unique, as the pyrene groups are easily released from glycopeptides during ionization when 2,5-dihydroxybenzoic acid is used as a matrix. As a result, most ions are observed as the underivatized form on the spectra. At the same time, pyrene derivatization dramatically reduces the ionization of peptides. Thus, for glycopeptides in a mixture of abundant peptides, we could obtain MS spectra in which the signals of glycopeptides were intense enough for subjection to MSn in order to determine the structures of both glycan and peptide. Finally, we show that the glycopeptides derived from as little as 1 ng of prostate specific antigen can be detected by this method. Glycoproteomics is a vital research field because it is estimated that over 70% of all human proteins are glycosylated.1 The glycans on proteins are significant because they play important biological roles in the body, including cell-cell interaction, cell recognition, and protein regulation. Although proteins are genetically encoded, their glycosylation state depends on the glycosylation-related enzymes that are present in the local cell environment. Changes in glycosylation are known to occur with certain diseases such as cancer. Thus, detection methods to monitor changes in the * Corresponding author. Phone: 81-3-3961-3255. Fax: 81-3-3964-5588. E-mail:
[email protected]. † Laboratory of Glycobiology. ‡ Laboratory of Glyco-Organic Chemistry. (1) Apweiler, R.; Hermjakob, H.; Sharon, N. Biochim. Biophys. Acta 1999, 1473, 4–8.
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glycosylation of glycoproteins are essential to search potential biomarkers for cancer and other diseases. However, investigation of the released glycans gives no information about the proteins from which the glycans originated. This disadvantage leads to serious problems in biomarker discovery. For example, it is not possible to identify whether or not the glycosylation profile has changed in a protein associated with the disease. In contrast to analyzing released glycans, glycopeptide analysis provides sitespecific glycosylation information on a protein, in addition to identification of the protein. This approach can potentially be useful for determining changes in glycosylation even though there may be no alteration in expression at the protein level. For the above reasons, development of methods for glycopeptide analysis is highly desirable. Currently, the methods applied to study glycopeptides (or glycoproteins) are most commonly based on MS techniques. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has several advantages over other techniques because it can rapidly and simultaneously detect many samples, and it can provide conclusive and reproducible evidence by the repetition of available measurements. In particular, spectrum interpretation is simple because MALDI of glycopeptides typically results in singly charged [M + H]+ or [M - H]- ions. As compared with nonglycosylated peptides, however, glycopeptides show lower ionization efficiency and the MS signals of glycopeptides are severely suppressed by those of nonglycosylated peptides. Moreover, most glycosylation sites carry a multitude of glycans, giving rise to different glycoforms. This phenomenon further reduces the relative amount of individual glycopeptides and makes their detection difficult. As a result, it is almost impossible to analyze glycopeptides without specific enrichment or separation procedures. For this purpose, lectin affinity chromatography is the most widely used technique.2,3 In general, with dependence on the lectin used, this method is biased toward particular structures due to binding specificity and is “targeted glycoproteomics” rather than “common glycoproteomics”. Another enrichment method using gel matrixes, such as cellulose or sepharose, is based on hy(2) Kubota, K.; Sato, Y.; Suzuki, Y.; Goto-Inoue, N.; Toda, T.; Suzuki, M.; Hisanaga, S.; Suzuki, A.; Endo, T. Anal. Chem. 2008, 80, 3693–3698. (3) Sparbier, K.; Wenzel, T.; Kostrzewa, M. J. Chromatogr., B 2006, 840, 29– 36. 10.1021/ac101555a 2010 American Chemical Society Published on Web 09/23/2010
drophilic interaction.4,5 However, many nonglycosylated peptides, such as those containing several hydrophilic amino acids, show as strong hydrophilicity as glycopeptides. Moreover, if glycans are attached to hydrophobic peptides or longer peptides, then they may not be retained by hydrophilic groups. Recently, diboronic acid-functionalized magnetic beads and boronic acid-mesoporous silica have been introduced for the relatively unbiased enrichment of glycopeptides.3,6,7 Nevertheless, the drawback remains that less abundant glycopeptides may not be easily detected among many other nonglycosylated peptides, which are still present even after such enrichment. In order to enhance the ion yield of oligosaccharides, the reducing end of the oligosaccharides can be derivatized with a reagent that has a hydrophobic group such as a fluorescent labeling reagent. For example, we previously succeeded in identifying subpicomole levels of isomeric oligosaccharides by MALDIMS after pyrene derivatization.8,9 As compared with other fluorescent reagents, pyrene derivatization gave superior mass spectra in terms of ion yield and S/N ratio in both the positive- and negative-ion modes. From this finding, hydrophobic derivatization with pyrene seems to improve both mixing with aromatic matrixes and the gas-phase ion production of oligosaccharides, as compared with underivatized oligosaccharides. As a new strategy to obtain intense signals of glycopeptides, we applied derivatization with another pyrene derivative, 1-pyrenyldiazomethane (PDAM), to glycopeptide samples without releasing glycans. PDAM is used as a fluorescent labeling agent for carboxylic acids10,11 and, at a minimum, the C-terminus of a peptide will be derivatized. PDAM readily reacts without catalysts and the products are stable at room temperature.10,11 In some cases, the acidic side chains of peptides and sialic acid residues of glycans should be modified. As expected, pyrene derivatization with PDAM increased the ionization of glycopeptides. At the same time, we found that the ionization of peptides was dramatically reduced, although both peptides and glycopeptides in the samples should be derivatized. Here, we describe an on-plate pyrene derivatization method using PDAM for highly sensitive MALDI-MSn of glycopeptides. EXPERIMENTAL SECTION Materials. Human serum albumin and bovine pancreatic ribonuclease B (RNaseB) were purchased from Sigma-Aldrich (Steinheim, Germany). Human prostate-specific antigen (PSA) from seminal fluid was obtained from Chemicon International (Billerica, MA). Trypsin Gold (Mass Spectrometry grade) and thermolysin were purchased from Promega (Madison, WI) and Calbio Chem (Merck, Darmstadt, Germany), respectively. ACTH fragment 18-39 was purchased from Sigma-Aldrich. For the MALDI matrix chemicals, purified 2,5-dihydroxybenzoic acid (4) Wada, Y.; Tajiri, M.; Yoshida, S. Anal. Chem. 2004, 76, 6560–6565. (5) Tajiri, M.; Yoshida, S.; Wada, Y. Glycobiology 2005, 15, 1332–1340. (6) Sparbier, K.; Koch, S.; Kessler, I.; Wenzel, T.; Kostrzewa, M. J. Biomol. Tech. 2005, 16, 407–413. (7) Xu, Y.; Wu, Z.; Zhang, L.; Lu, H.; Yang, P.; Webley, P. A.; Zhao, D. Anal. Chem. 2009, 81, 503–508. (8) Amano, J.; Sugahara, D.; Osumi, K.; Tanaka, K. Glycobiology 2009, 19, 529–600. (9) Amano, J.; Osanai, M.; Orita, T.; Sugahara, D.; Osumi, K. Glycobiology 2009, 19, 601–614. (10) Nimura, N.; Kinoshita, T.; Yoshida, T.; Uetake, A.; Nakai, C. Anal. Chem. 1988, 60, 2067–2070. (11) Schneede, J.; Ueland, P. M. Anal. Chem. 1992, 64, 315–319.
Figure 1. (A) Structures of glycopeptides 1 and 2 (GP-1 and GP2). Open circle, galactose; closed circle, mannose; square, Nacetylglucosamine; diamond, N-acetylneuraminic acid. (B) Reaction path of PDAM with carboxylic acid.
(DHBA) and R-cyano-4-hydroxycinnamic acid (CHCA), were purchased from Shimadzu-Biotech (Kyoto, Japan); and 3-aminoquinoline (3AQ) was purchased from Sigma-Aldrich. 1-Pyrenyldiazomethane (PDAM) was purchased from Molecular Probes, Inc. (Eugene, OR). Acetonitrile (LC/MS grade), ethanol (LC/ MS grade), 1-butanol (HPLC grade), and trifluoroacetic acid (TFA, HPLC grade) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Preparation of Glycopeptide-1 (GP-1) and GP-2. As shown in Figure 1, the sialylglycopeptide GP-2 was prepared from hen egg yolk.12 GP-2 was then converted to GP-1 (Figure 1), which contains the prostate-specific antigen (PSA) fragment 59-63, IRNKS. In order to separate the product from the reaction mixture, the three amino groups of GP-2 were acetylated with NaHCO3 and N-acetoxysuccinimide in acetone-water (1:1) at room temperature for 90 min. Tri-N-Ac-GP-2 was purified by HPLC using a preparative ODS column. Peptide IRNKS with a GlcNAc residue was prepared by an Fmoc solid-phase method using Fmoc-Asn(GlcNAc(OAc)3)-OH.13,14 The GlcNAc(OAc)3-peptide was obtained by treatment with TFA containing 2.5% triisopropylsilane and 2.5% water and purified using an ODS column. The O-Ac groups of the GlcNAc moiety were removed with aqueous tetrabutylammonium hydroxide, and IRN(GlcNAc)KS was obtained. For transglycosylation, tri-N-Ac-GP-2 as a glycoside donor and IRN(GlcNAc)KS as an acceptor were incubated with recombinant Endo-M (Y217F)15 in phosphate buffer pH 6.6 at 30 °C for 18 h. Sialylated GP-1 was purified using a preparative ODS column. GP-1 was obtained after desialylation by heating in 0.8% TFA. Digestion of Glycoproteins or Protein. RNaseB or albumin was incubated in 10 mM ammonium bicarbonate containing 10 mM dithiothreitol at 55 °C for 45 min. After cooling, 5 µL of 135 mM iodoacetamide was added to the mixture, which was then kept in the dark for 45 min. The mixture was heated at 100 °C for 5 min after adding RapiGest SF (Waters; Milford, MA) to a final concentration of 0.1% in 50 mM ammonium bicarbonate. After the (12) Seko, A.; Koketsu, M.; Nishizono, M.; Enoki, Y.; Ibrahim, H. R.; Juneja, L. R.; Kim, M.; Yamamoto, T. Biochim. Biophys. Acta 1997, 1335, 23–32. (13) Mizuno, M.; Muramoto, I.; Kobayashi, K.; Yaginuma, H.; Inazu, T. Synthesis 1999, 162–165. (14) Inazu, T.; Kobayashi, K. Synlett 1993, 869–870. (15) Umekawa, M.; Huang, W.; Li, B.; Fujita, K.; Ashida, H.; Wang, L.-X.; Yamamoto, K. J. Biol. Chem. 2008, 283, 4469–4479.
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Figure 2. Positive-MS of GP-1 (5 fmol) after PDAM derivatization using a DHBA matrix (A), and negative-MS of GP-1 (10 fmol) after PDAM derivatization using a DHBA matrix (B). Positive-MS (C) and negative-MS (D) of the same samples without derivatization. MS2 spectra of the protonated ion at m/z 2454 (E) and the deprotonated ion at m/z 2452 (F).
mixture was cooled, it was incubated with 1 µg of trypsin at 37 °C overnight. The tryptic digest of albumin was desalted using a PepClean C-18 Spin Column (Pierce, Rockford, IL), and the digest of RNase B was subjected to enrichment by hydrophilic interaction using cellulose fibros medium.4 The fractions were dried on a Speed Vac. Pyrene Derivatization on the Target Plate. Each sample solution (0.5 µL) for analysis was placed on a target plate and dried in air. A fresh solution of PDAM at a concentration of 500 or 2500 pmol/0.25 µL in dimethyl sulfoxide was added to the dried sample, and the plate was incubated at 80 °C until dry. The plate was rinsed with toluene to remove excess PDAM and dried. Mass Spectrometry. As the matrix, 2,5-dihydroxybenzoic acid (DHBA) in 60% acetonitrile (10 mg/mL) was added to dried samples with or without pyrene derivatization on the plate. In some cases, a liquid matrix (3AQ-CHCA) was used instead. A stock solution of 3AQ-CHCA was prepared by dissolving 35 mg of 3AQ in 150 µL of a saturated solution of CHCA in MeOH. A 10 µL aliquot of the 3AQ-CHCA stock solution was diluted with 90 µL of 60% acetonitrile. The mixture was analyzed with two kinds of MALDI-TOFMS, AXIMA-QIT or AXIMA-Performance (Shimadzu Biotech, Kyoto, Japan). Analysis of the Digest of PSA. PSA (50 ng) was incubated with 10 U of thermolysin in 10 µL of 50 mM ammonium bicarbonate, pH 8, at 56 °C for 16 h, and the mixture was then dried on a Speed Vac. For desialylation, the thermolysin-digested samples were heated in 0.8% TFA at 80 °C for 45 min and dried on a Speed Vac. The dried digest was dissolved in water, and an aliquot was directly analyzed with MALDI-QIT-TOFMS after derivatization with PDAM. RESULTS AND DISCUSSION The carboxylic acid groups of glycopeptides were directly derivatized by PDAM on the MALDI plate and converted into the ester (Figure 1) because the reaction proceeds readily without catalysts.10,11 In this way, the acidic side chains and C-terminus of the peptide should be modified, as well as the sialic acid residues of glycans. Therefore, glycopeptides with both neutral and sialylated glycans can be derivatized. As shown in Figure 8740
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2A,B, after derivatization 5 or 10 fmol of GP-1 was satisfactorily detected in both positive- and negative-MALDI-QIT-TOFMS using DHBA as a matrix, although poor signals were obtained without PDAM treatment (Figure 2C,D). Contrary to our expectations, only the underivatized ion (m/z 2240 and 2238 in parts A and B of Figure 2, respectively) was observed in the spectrum along with some minor peaks corresponding to the ion derivatized with pyrene (m/z 2454 and 2452 in parts A and B of Figure 2, respectively). The same results were obtained when the reaction time was prolonged or the amount of PDAM increased (data not shown). We compared the spectra obtained by MALDI-QITTOFMS to those obtained by the linear mode of MALDI-TOFMS. The results using DHBA as a matrix were identical (Supplementary Figure 1A,B in the Supporting Information). With the use of the liquid matrix 3AQ-CHCA, however, the derivatized ion was mainly detected (Supplementary Figure 1C in the Supporting Information). These results strongly indicate that GP-1 was completely derivatized, but the ester-bond was subsequently cleaved by in-source decay using DHBA and the underivatized form was ultimately obtained. Intriguingly, substituting PDAM with 1-pyrenylmethanol, which does not react with glycopeptide, did not enhance ionization (data not shown). This finding indicates that the derivatization is likely to be essential for enhancing the signal. Therefore, the on-plate PDAM derivatization is unique. During ionization, the pyrene group is easily dissociated from glycopeptides, and in fact glycopeptides can be detected as the underivatized form. Positive-MS2 analysis of the derivatized ion at m/z 2454 demonstrated that pyrene had been introduced to the peptide (Figure 2E). The protonated ion at m/z 914 (IRNKS + 214 + 83 + 1) consisted of the pyrene-labeled peptide and the fragment obtained by ring cleavage of the GlcNAc residue, whereas the protonated ion at m/z 1034 (IRNKS + 214 + 203 + 1) was the pyrene-labeled peptide with GlcNAc. By contrast, the negativeMS2 spectrum of the deprotonated ion at m/z 2452 showed only the 0,2X ion at m/z 912 (IRNKS + 214 + 83 - 1) and not the Y ion at m/z 1032 (IRNKS + 214 + 203 - 1), in addition to two glycan fragments, the 2,4A ions at m/z 1275 and 1478
Figure 3. Negative-MS of glycopeptides prepared from RNase B by trypsin digestion and derivatization with PDAM (A) and MS2 of the ion at m/z 1689 (C). Negative-MS of glycopeptides without derivatization did not show any signals of corresponding glycopeptides (B).
(Figure 2F, fragmentation nomenclature by Domon and Costello16). These glycan fragment ions observed in negative mode are obtained from both underivatized and derivatized forms and are useful for the determination of glycan structures, as described later. The on-plate PDAM derivatization is also effective at enhancing ionization of sialylated glycopeptides. Furthermore, another advantage of PDAM derivatization is an increase in the stability of sialic acid residues. Negative-MS of GP-2 without PDAM derivatization detected only the desialylated form (m/z 2282) with poor signal strength and no sialylated derivative (Supplementary Figure 2A in the Supporting Information). After derivatization, however, ionization was markedly improved and a glycopeptide ion at m/z 3292 was identified in which all sialic acid residues could be found (Supplementary Figure 2B in the Supporting Information). Despite the presence of three carboxylic acids groups, only two pyrene groups were detected on a molecule. This is because in-source decay was not entirely suppressed using 3AQCHCA as a matrix and the ion at m/z 3292 which had one free carboxylic acid resulted in easy detection as a negative ion. Nonetheless, this kind of ion is useful for determining the sialyl linkage. Indeed, we have succeeded in discrimination of isomeric sialylated glycopeptides by MALDI-MS2 using 3AQ-CHCA after PDAM derivatization (manuscript submitted for publication). Next, we applied this method to the analysis of glycopeptides prepared from RNase B. After PDAM derivatization, the glycopeptide ions at m/z 1690, 1852, 2014, 2176, and 2338, corresponding to the peptide NLTK with Man5, Man6, Man7, Man8, and Man9 glycans, respectively, were readily detected in negativeMS (Figure 3A), whereas the same analyte without PDAM derivatization did not show any glycopeptide signals (Figure 3B). In negative-MS2 of the ion at m/z 1689, the ions at m/z 1072 and 869, corresponding to glycan fragment ions, and the ion at m/z 556, corresponding to [peptide + 83 - H]-, were observed (Figure 3C). The ions at m/z 1645 (1689 - 44) and 512 (556 - 44), which result from loss of CH3CHO, suggested the (16) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397–409.
presence of threonine.17 In negative-MS3 of the ion at m/z 1072, the D, A, C, and Y ions were obtained and the Man5 glycan was identified, as shown in Figure 4A. Negative-MS3 of the ion at m/z 556 in Figure 3C confirmed that this was peptide NLTK with a glycan fragment on the asparagine (Figure 4B). As described above we found that, as well as the increase in glycopeptide ion abundance, the peptide ions of RNase B dramatically decreased after PDAM derivatization. As a result, the effects of PDAM on nonglycosylated peptides were investigated because PDAM should react with all kinds of peptides. A mixture of ACTH18-39 peptide (2 pmol) and GP-1 (50 fmol) was measured in positive- and negative-MS with or without PDAM derivatization on the plate. As expected, only peptide ion (m/z 2465 or 2463) and not GP-1 was detected in the underivatized mixture because of ion suppression by the peptide (Figure 5A,B); after PDAM derivatization, however, an intense signal corresponding to the GP-1 ion was obtained in both positive and negative modes (Figure 5C,D). Next, the effects of using different amounts of PDAM on the extent of ionization of peptides or glycopeptides were investigated. A mixture of nonglycosylated peptides was prepared by trypsin digestion of human serum albumin, which has no glycan, leading to more than 10 kinds of peptides. When GP-1 (1 ng) was added to the albumin peptides (100 ng), there was no signal corresponding to GP-1 in the absence of PDAM (Figure 6A). Derivatization of the same mixture with PDAM (500 pmol), however, resulted in a GP-1 signal, instead of a decrease in peptide signal. Furthermore, derivatization with 2.5 nmol of PDAM resulted in the same intensity of GP-1 signal but a reduced peptide signal. Also in the case of glycopeptides from RNase B (Figure 6B), PDAM derivatization simultaneously enhanced glycopeptide ionization and suppressed nonglycosylated peptide ionization. These results show that ionization of the albumin peptides, which are listed in Table 1 and have different properties in terms of acidity, hydrophobicity, and aromaticity, decreased depending on the (17) Bowie, J. H.; Brinkworth, C. S.; Dua, S. Mass Spectrom. Rev. 2002, 21, 87–107.
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Figure 4. Negative-MS3 of the ion at m/z 1072 (A) and the ion at m/z 556 (B) in Figure 3C.
Figure 5. Positive-MS of a sample containing GP-1 (50 fmol) and ATCH18-39 peptide (2 pmol) without (A) or with (C) pyrene derivatization. Negative-MS of the same sample without (B) or with (D) pyrene derivatization.
amount of PDAM used. The same effect could be seen for mixtures containing a reduced level of GP-1, e.g., same experiment using 100-fold less GP-1 (10 pg) or increasing the amount of albumin peptides (200 ng). A sample containing only 0.2% of glycopeptides produced a signal sufficient for subjection to MS2 analysis, and even as little as 0.1% of GP-1 added to the peptides could be conclusively detected. Peptides, including those derived from albumin and ATCH, which have varying numbers of carboxylic acids, differences in hydrophobicity and aromaticity, and pI values ranging from 4.3 to 9.8, as shown in Table 1, reduced ionization after PDAM derivatization, although the extent of this reduction differed. For glycopeptides that had longer or more hydrophobic peptides, PDAM derivatization had little effect because these signals were strong even in the absence of PDAM. The most dramatic effect was obtained for glycopeptides with a short peptide compared with 8742
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Figure 6. Relationship between signal intensity and amount of derivative reagent used. (A) Relative signal intensities were obtained by positive-MS of a sample containing GP-1 (1 ng) and peptides from human serum albumin (100 ng) obtained by trypsin digestion followed by derivatization with PDAM at 0, 500 pmol, or 2.5 nmol: circle, ion at m/z 960; square, ion at m/z 1139; diamond, ion at m/z 1372; triangle, ion at m/z 1444; reverse triangle, ion at m/z 1640; closed circle with broken line, GP-1 ion at m/z 2240. The values of the GP-1 ion at m/z 2240 in the absence of peptides are also indicated (closed circle with solid line). B, Relative signal intensities were obtained by negative-MS of a sample containing glycopeptides (1 ng) from bovine RNaseB and peptides from human serum albumin (100 ng) obtained by trypsin digestion followed by derivatization with PDAM at 0, 500 pmol or 2.5 nmol: circle, ion at m/z 1147; square, ion at m/z 1442; diamond, ion at m/z 1638; triangle, ion at m/z 1909; reverse triangle, ion at m/z 2043; closed circle with broken line, glycopeptide ion at m/z 1689. The values of the glycopeptide ion at m/z 1689 in the absence of peptides are also indicated (closed circle with solid line).
the glycan part of the molecule, where ionization is strongly suppressed in the presence of peptides. Therefore, the selection of protease is important. Different kinds of proteases should be used for a sample containing unknown glycoproteins. One reason for the selective ionization of glycopeptides but not peptides may be that the introduction of a pyrene group enhances the mixing efficiency between the matrix and more hydrophilic glycopeptides by increasing hydrophobicity. By contrast, peptides attached to a pyrene group are more hydrophobic, which may lead to incomplete mixing in the matrix solution. Finally, we could directly detect glycopeptides in a sample of 1 ng of PSA digested by thermolysin without any purification or
Table 1. Properties of Peptides Used in This Study
a
name
monoisotopic mass
no. of residue
sequencea
pI
ACTH 18-39 Albumin 427-434 Albumin 500-508 Albumin 66-75 Albumin 187-198 Albumin 287-298 Albumin 414-426 Albumin 509-524
2464.2 959.6 1137.5 1148.6 1370.6 1442.6 1638.7 1909.9
22 8 9 10 12 12 13 16
RPVKVYPNGAEDESAEAFPLEF FQNALLVR C*C*TESLVNR LVNEVTEFAK AAFTEC*C*QAADK YIC*ENQDSISSK QNC*ELFEQLGEYK RPC*FSALEVDETYVPK
4.3 9.8 6.0 4.5 4.4 4.4 4.3 4.7
C* indicates carbamidomethylated Cys.
Figure 7. Direct MS analysis of a reaction mixture of PSA (1 ng) digested with thermolysin. Negative-MS of the mixture without any purification was measured before (A) or after (B) on-plate pyrene derivatization.
enrichment. One of the glycopeptides produced from PSA by thermolysin had the same structure of GP-1 and accounted for approximately 6% of the molecule. PDAM derivatization led to the successful detection of two kinds of glycopeptides, namely, GP-1 (m/z 2240) and fucosylated GP-1 (m/z 2386), whereas no glycopeptide signals were seen without PDAM derivatization (Figure 7). CONCLUSIONS In the present work, we have developed a new derivatization method that is suitable for sensitive MALDI-MS of glycopeptides even in the presence of an abundance of nonglycosylated peptides. The new method is effective enough for glycopeptides alone, but it is also effective for glycopeptide samples in which peptides still remain after enrichment. The method provides better yields of both positive- and negative-ions from glycopeptides as compared with underivatized samples. Furthermore, pyrene derivatization
dramatically diminishes the ionization of peptides. As a result of pyrene derivatization, conventional MS spectra that show only peptide signals can be converted to new spectra revealing strong signals of glycopeptides. Pyrene derivatization led to enhanced signals of glycopeptides present in very small amounts (10 pg) or in very low relative abundance (0.1%). This simple and rapid method involves the addition of a pyrene derivative to analytical samples on the target plate prior to MALDI-MS and is complete within 30 min. We believe this novel technique represents a significant advance in glycoproteomics. Interestingly, the pyrene group can be released by in-source decay using DHBA as a matrix and, as a result, underivatized molecules are detected. This is an advantage because it means that spectrum interpretation is simple and common databases can be applied. Another advantage of this technique is that when using the liquid matrix, 3AQ-CHCA, the sialyl linkages of pyrenesialylated glycopeptides are stable on MSn due to the inefficient release of the pyrene group. Thus, the fragmentation obtained will provide more detailed structures as compared with conventional MSn, which shows only desialylated products because of the preferential detachment of the sialic acid residue by in-source decay or collision-induced decay. In short, PDAM derivatization is a novel type of derivatization that can change its application according to the chosen matrix. ACKNOWLEDGMENT This work is supported in part by SENTAN, JST (Japan Science and Technology Agency). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 11, 2010. Accepted September 9, 2010. AC101555A
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