Anal. Chem. 2006, 78, 1093-1103
Determination and Characterization of Site-Specific N-Glycosylation Using MALDI-Qq-TOF Tandem Mass Spectrometry: Case Study with a Plant Protease Natalia V. Bykova,*,† Christof Rampitsch,† Oleg Krokhin,‡ Kenneth G. Standing,‡ and Werner Ens‡
Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba R3T 2M9, Canada, and Department of Physics and Astronomy, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
MALDI tandem mass spectrometry analysis on a hybrid quadrupole-quadrupole time-of-flight (Qq-TOF) instrument was used in combination with two-dimensional gel electrophoresis, proteolytic digestion, and liquid chromatography for identification and structural characterization of glycosylation in a novel glycoprotein, pathogenesisrelated subtilisin-like proteinase P69B from tomato. Glycopeptide fractions from microcolumn reversed-phase HPLC deposited on MALDI targets were identified from MS by their specific m/z spacing patterns (203, 162, 146 u) between glycoforms. In most cases, MS/MS spectra of [M + H]+ ions of glycopeptides featured peaks useful for determining sugar compositions, peptide sequences, and thus probable glycosylation sites. Furthermore, peptiderelated product ions could readily be used in database search procedures to identify the glycoprotein. Four out of five predicted glycosylation sites were biologically relevant and occupied by five N-linked glycan side chains each. In addition, the fragmentation efficiency allowed detection of further modification of methionine-containing glycoforms with either oxidized or iodoacetamide alkylated methionine. The high resolution furnished by MALDI-QqTOF allowed rapid and sensitive structural characterization of site-specific N-glycosylation from a limited quantity of material and revealed heterogeneity at different levels, including different glycan side-chain modifications, and heterogeneity of oligosaccharide structures on the same glycosylation site. N-Glycosylation is one of the most common posttranslational modifications of proteins; it is formed by covalent attachment of asparagine-linked carbohydrates to the parent protein. The Nlinked glycans have received attention from various analytical methods, but no single technique has been capable of providing a complete structural analysis, because of the very large number of possible structural isomers. Techniques based on mass spectrometry (MS) for the analysis of glycans have been directly interfaced with proteomics. They yield the most information, such * Corresponding author. Tel: (204) 983-1465. Fax: (204) 983-4604. E-mail:
[email protected]. † Agriculture and Agri-Food Canada. ‡ University of Manitoba. 10.1021/ac0512711 CCC: $33.50 Published on Web 01/06/2006
© 2006 American Chemical Society
as determination of glycosylation sites, structures and amounts of each glycan at each site, occupancy at each site, and the number of individual glycoforms,1 although the last step is rarely attempted because of the complexity of the mixtures of possible compounds. MS analysis of gel-separated proteins is particularly useful to study species, tissue, and cell type-specific protein glycosylation, where only small quantities of naturally expressed protein mixtures need to be separated.2 Considering the extensive heterogeneity of individual glycosylation sites, this analytical strategy is especially desirable, since it is highly sensitive and at the same time rapid and easily accessible to the broad proteomics community. Structural determination of the individual glycans is much more complicated than for other biopolymers on account of the branched and isomeric nature of the glycans.3 The use of an appropriate ionization technique, the extent of fragmentation, sensitive detection of diagnostically important fragment ions, and accuracy of mass measurements have been postulated as major determinants for the overall success of MS-based methodologies.4 Most common mass spectrometric approaches to characterize glycoproteins are based on a chemical or enzymatic glycan release followed by labeling, separation by chromatography or electrophoresis, and detection in MS or MS/MS mode. However, in these approaches, information about glycosylation site occupancy and heterogeneity is often lost. Protein glycosylation should ideally be characterized directly at the glycopeptide level. Until recently, glycopeptides were mostly analyzed by MALDI-TOF MS or by electrospray ionization (ESI) MS. Further collision-induced dissociation (CID) and tandem mass spectrometry analysis was performed using electrospray instruments with a quadrupole and a TOF analyzer. This approach was more successful for localization of small O-glycans5 but for N-glycosylated peptides mostly provided information on the glycan moiety with dominating fragment ions resulting from the cleavage of glycosidic linkages and lack peptide fragmentation ions.6 Under ESI conditions, specific mass spectral detection of glycopeptides from a protein (1) Harvey, D. J. Proteomics 2001, 1, 311-328. (2) Ku ¨ ster, B.; Krogh, T. N.; Mørtz, E.; Harvey, D. J. Proteomics 2001, 1, 350361. (3) Laine, R. A. Glycobiology 1994, 4, 759-767. (4) Mechref, Y.; Novotny, M. V.; Krishnan, C. Anal. Chem. 2003, 75, 48954903. (5) Macek, B.; Hofsteenge, J.; Peter-Katalinic, J. Rapid Commun. Mass Spectrom. 2001, 15, 771-777.
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digest during on-line HPLC-MS was achieved with either selected ion monitoring for diagnostic product ions generated by cone voltage fragmentation7 or precursor ion scanning for reporter oxonium fragment ions of monosaccharides8 and much larger glycoforms.9 Although the lability of the glycosidic bond is favorable for sensitive and selective detection of glycopeptides by precursor ion scanning, it affects the fragmentation of the peptide backbone negatively, showing lower yields as a secondary fragmentation process. To induce peptide backbone cleavage to derive peptide sequence information, fairly harsh CID conditions are required.6 With ESI-MS, the most useful information in terms of unambiguous sequence determination and localization of glycosylation sites has been obtained by performing MS/MS on [peptide + GlcNAc]+ ions obtained by in-source fragmentation of the corresponding glycopeptides and at high declustering voltage values.10 ESI-quadrupole ion trap mass spectrometry (ESI-QIT-MS), with its capacity to perform multiple stages of fragmentation (MSn), was demonstrated as an effective method for the structural characterization of complex glycoprotein oligosaccharides.11 For determination of glycopeptide structures, application of MSn provided by ion trap instruments allowed trimming of the glycan moiety in an MS/MS step down to a single GlcNAc, which was further fragmented in MS3 analysis and revealed information on the peptide sequence and the glycan attachment site.12,13 Recently, Demelbauer et al. demonstrated that the necessity of multistage CID experiments for complete structural elucidation could be overcome by application of MALDI-QIT reflectron TOF instrument (QIT-rTOF), which yielded both kinds of fragments at the MS2 stage without mutual interference.14 Electron-capture induced dissociation (ECD) using Fourier transform mass spectrometry (FT-MS) has been introduced to characterize N-glycosylated peptides from an unfractionated tryptic protein digest.15 ECD provided c and z• ions, revealing many peptide bond cleavages with retention of the intact glycan moiety. Further application of another dissociation technique, infrared multiphoton dissociation (IRMPD), in FT-MS provided abundant fragment ions, primarily through dissociation at glycosidic linkages. In contrast to ECD, the IRMPD spectrum provided no information about the peptide fragmentation. The two types of spectra yielded complementary peptide and oligosaccharide sequence information. The development of MALDI-TOF/TOF analyzers has been described for the detection of peptides16 and oligosaccharides4 and carries the potential benefits of producing high-energy
fragmentation. The spectra exhibit high numbers of cross-ring cleavages instead of the predominat series of glycosidic cleavages obtained under low-energy CID conditions,17 which provide important information on the stereochemistry of individual sugar residues, the linkage position, and branching structure.18 Structural characterization of N-glycopeptides has recently been demonstrated using MALDI-TOF/TOF instrument equipped with the LIFT-MS/MS facility for acceleration of fragment ions generated by laser-induced decomposition of precursor ions.19 This technique resulted in fragmentation of both peptide and glycan moieties of glycopeptides. The peptide sequence tags could be derived from y-type and in some cases b-type of cleavage, whereas the y-type ions comprising the potential N-glycosylation sites exhibited complete N-glycan structures. Fragmentation of the glycan moiety resulted in cleavages of glycosidic bonds as well as a ring cleavage of the innermost GlcNAc. Similar instrumentation was used in another study to characterize matrix-dependent selective fragmentation of oligosaccharides and N-glycopeptides.20 In this case, when 2,5-dihydroxybenzoic acid (DHB) matrix was employed, fragmentation of N-glycosylated hexapeptide occurred predominantly at the glycoside bond, resulting in two major product ions due to the monoglycosylated peptide ion and the hexasaccharide ion. A series of b- and y-ions were also useful for peptide sequencing and assignment of glycosylation sites. In addition, sodiated precursor ion of N-glycopeptide derived in the presence of R-cyano-4-hydroxycinnaminic acid (CHCA) matrix produced glycoside bond fragmentation with minimal cleavage of the peptide backbone, which allowed determination of sugar chains. Quadrupole-orthogonal time-of-flight (QqTOF) type instruments with a MALDI ionization source21,22 have improved precursor ion selection and substantially higher product ion resolution than with postsource decay (MALDI-PSD TOF).23 The use of MALDI-QqTOF for tandem MS of oligosaccharides was shown to be particularly attractive, and it was clearly demonstrated that this geometry is sensitive enough to allow analysis of oligosaccharides released from SDS-PAGE spots.24 Useful data were obtained recently from MALDI-Qq-TOF MS/MS analysis of glycopeptide [M + H]+ ions that featured consistent fragmentation pattern and provided both oligosaccharide and peptide sequence information.25,26 In the present study, MALDI-Qq-TOF tandem MS was employed to elucidate the glycans’ structure and their specific sites of attachment in a native glycoprotein without prior enzymatic release. We describe de novo detection and characterization of
(6) Jebanathirajah, J.; Steen, H.; Roepstorff, P. Am. Soc. Mass Spectrom. 2003, 14, 777-784. (7) Medzihradszky, K. F.; Maltby, D. A.; Berger, F. G.; Baumann, H. Am. Soc. Mass Spectrom. 1994, 5, 350-358. (8) Carr, S.; Huddleston, M.; Bean, M. Protein Sci. 1993, 2, 183-196. (9) Ritchie, M. A.; Gill, A. C. Am. Soc. Mass Spectrom. 2002, 13, 1065-1077. (10) Hui, J. P. M.; White, T. C.; Thibault, P. Glycobiology 2002, 12, 837-849. (11) Weiskopf, A. S.; Vouros, P.; Harvey, D. J. Anal. Chem. 1998, 70, 44414447. (12) Wada, Y.; Tajiri, M.; Yoshida, S. Anal. Chem. 2004, 76, 6560-6565. (13) Wuhrer, M.; Koeleman, C. A. M.; Hokke, C. H.; Deelder, A. M. Anal. Chem. 2005, 77, 886-894. (14) Demelbauer, U. M.; Zehl, M.; Plematl, A.; Allmaier, G.; Rizzi, A. Rapid Commun. Mass Spectrom. 2004, 18, 1575-1582. (15) Håkansson, K.; Cooper, H. J.; Emmett, M. R.; Costello, C. E.; Marshall, A. G.; Nilsson, C. L. Anal. Chem. 2001, 73, 4530-4536. (16) Medzihradszky, K. F.; Campbell, J. M.; Baldwin, M. A.; Falick, A. M.; Juhasz, P.; Vestal, M. L.; Burlingame, A. L. Anal. Chem. 2000, 72, 552-558.
(17) Lewandrowski, U.; Resemann, A.; Sickmann, A. Anal. Chem. 2005, 77, 3274-3283. (18) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227. (19) Wuhrer, M.; Hokke, C. H.; Deelder, A. M. Rapid Commun. Mass Spectrom. 2004, 18, 1741-1748. (20) Kurogochi, M.; Nishimura, S.-I. Anal. Chem. 2004, 76, 6097-6101. (21) Krutchinsky, A. N.; Loboda, A. V.; Spicer, V. L.; Dworschak, R.; Ens, W.; Standing, K. G. Rapid Commun. Mass Spectrom. 1998, 12, 508-518. (22) Loboda, A. V.; Krutchinsky, A. N.; Bromirski, M.; Ens, W.; Standing, K. G. Rapid Commun. Mass Spectrom. 2000, 14, 1047-1057. (23) Harvey, D. J. J. Am. Soc. Mass Spectrom. 2000, 11, 572-577. (24) Hanrahan, S.; Charlwood, J.; Tyldesley, R.; Langridge, J.; Bordoli, R.; Bateman, R.; Camilleri, P. Rapid Commun. Mass Spectrom. 2001, 15, 11411151. (25) Krokhin, O.; Ens, W.; Standing, K. G.; Wilkins, J.; Perreault, H. Rapid Commun. Mass Spectrom. 2004, 18, 1-12. (26) Ethier, M.; Krokhin, O.; Ens, W.; Standing, K. G.; Perreault, H. Rapid Commun. Mass Spectrom. 2005, 17, 2713-2720.
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four N-glycosylation sites in tomato pathogenesis-induced subtilisin-like P69B protease with site-specific distribution of glycoforms, using a combination of two-dimensional gel electrophoresis, proteolytic digestion, liquid chromatography, and MALDI-Qq-TOF tandem mass spectrometry. EXPERIMENTAL SECTION Materials. Tomato (Lycopersicon esculentum) cultivar Bonny Best was grown in pots at 20 °C, 60% relative humidity, under 16-h-light/8-h-dark cycle. Briefly, organellar-free fluids were prepared by grinding fresh leaves from eight-week-old plants using a Polytron (2 × 10 s bursts) and ice-cold grinding medium containing 10 mM MOPS/KOH, pH 7.2, 0.3 M mannitol, 0.5% (w/v) poly(vinylpolypyrrolidone), 5 mM dithiothreitol (DTT), 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin, and phosphatase inhibitor cocktails (inhibitor 1 and inhibitor 2; SigmaAldrich), with the grinding medium to material mass ratio 5:1. The homogenate was filtered through nylon cloth (120 µm) and centrifuged at 1000g for 7 min. The supernatant was centrifuged at 20000g for 20 min, and the organellar pellet was discarded. To remove small membrane particles, the supernatant was further centrifuged at 90000g for 1 h and then concentrated using 5-kDa cutoff filter concentrators (Vivascience Inc.). Preparative Two-Dimensional Isoelectric Focusing (IEF)/ SDS-PAGE. IEF was conducted with the Multiphor II system (Amersham Biosciences Inc., Piscataway, NJ) using 13-cm ReadyStrip IPG strips with a linear pH gradient range 4-7 according to the manufacturer’s instructions.27 Proteins (200 µg) were solubilized in 250 µL of rehydration solution containing 9 M urea, 20 mM DTT, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 0.2% (w/v) ampholyte (Biolyte 3-10, BioRad Laboratory, Mississauga, ON, Canada). The second dimension Tris-glycine SDS-PAGE was carried out using 12% acrylamide gels as described.28 After electrophoresis, gels were fixed in 12% (w/v) trichloroacetic acid for 1 h, stained overnight with 1% (w/v) Coomassie Blue R250, and destained for 2 h with 10% (v/v) ethanol, 7% (v/v) acetic acid. In Situ Digestion, HPLC Separation, Fraction Collection, and Deposition. For in-gel digestion, protein spots were excised from 2D gels cubed to 1 mm3, washed with 100 mM NH4HCO3, reduced with 10 mM DTT for 45 min at 56 °C, alkylated with 55 mM iodoacetamide for 30 min in the dark at room temperature, washed again with 100 mM NH4HCO3, and digested overnight at 37 °C with modified trypsin (Promega, sequencing-grade). The resulting tryptic peptides were extracted from the gel pieces by increasing concentration of acetonitrile (0, 5, 60, and 90%) in the presence of 5% formic acid in zero acetonitrile solution and 1% formic acid in other solutions. The peptide extracts were dried down in a SpeedVac centrifuge and dissolved in 0.1% trifluoroacetic acid (TFA) aqueous solution. The mixture was injected into a µ-HPLC system (micro-Agilent 1100 series, Agilent Technologies, Wilmington, DE). Samples were injected directly onto a 150 µm × 100 mm column with C18 material (Vydac 218 TP C18, 5 µm; Grace Vydac, Hesperia, CA) packed using a high-pressure vessel. Peptides were eluted with a linear gradient of 0-80% acetonitrile containing 0.1% TFA in 40 min at 4 µL/min flow rate. The column effluent was mixed online with DHB (150 mg/mL in water/acetonitrile 1:1) matrix solution (supplied at 0.5 µL/min) and deposited by a computer-
controlled robot25 onto a movable gold MALDI target at 1-min intervals. A Microtee P775 (Upchurch Scientific) was used for online mixing in the microflow version. Fractions were air-dried and subjected to MALDI-MS analysis. MALDI-Qq-TOF MS/MS Analysis of Tryptic Peptides. The chromatographic fractions were analyzed by single mass spectrometry (MS) with m/z range 560-5000 and by tandem mass spectrometry (MS/MS) analysis using the Manitoba/Sciex prototype QqTOF mass spectrometer.22 In this instrument, ions are produced by irradiation of the target with photon pulses from a 10-20-Hz nitrogen laser (VSL337ND, Spectra-Physics, Mountain View, CA) with 300 µJ energy/pulse. Orthogonal injection of ions from the quadrupole into the TOF section normally produces a mass resolving power of 10 000 full width at half-maximum and accuracy within a few millidaltons in the TOF spectra in both MS and MS/MS modes. Argon was used as the cooling gas in q0 (preanalyzer quadrupole) and as the collision gas in q2 (collision cell). During low-energy CID MS/MS, positive ions exiting from the first quadrupole are accelerated by a drop in voltage before entering the collision cell. The extent of voltage drop depends on the m/z of the peptide considered and was ∼50 V/1000 Da in this case. Because the maximum value of the voltage drop is 160 V, large analytes could not be fragmented efficiently over the whole m/z range. Thus, for precursor ions with m/z >3500, it was difficult to observe significant peptide fragmentation in MS/MS spectra. Peak Assignments and Identification of N-Glycopeptides. MALDI-MS/MS spectra were interpreted using Mascot MS-MS Ions Search (Matrix Science Ltd., London, U.K.) and Sonar MS/MS (Proteometrics Ltd., New York, NY) search engines using the nonredundant NCBI protein database. GlycoMod tool (www.expasy.org) was used to facilitate calculation of possible oligosaccharide compositions to given molecular ions.29 The tandem mass spectra derived from fragmentation of N-glycopeptides were inspected manually and had a characteristic signature pattern, i.e., the quadruplet [(peptide - 17) + H]+, [peptide + H]+, [(peptide + GlcNAc) + H]+, and [(peptide + CHCHNHAc + H]+ ion peaks. The [peptide + H]+ m/z value was used in search engines as a precursor ion for the peptide part of the spectra, together with the m/z values of peptide product ions in the mass spectra regions below the [peptide + H]+ ions. RESULTS AND DISCUSSION Detection and Characterization of Glycoforms. Total soluble proteins from tomato leaf organellar-free fluids were separated by two-dimensional isoelectric focusing/SDS-PAGE with Coomassie blue staining. Subtilisin-like P69B proteinase was identified by mass spectrometry in several spots and in a smear with an apparent pI 5.5-6.5, migrating at MW ∼70K (Figure 1). The two largest and most alkaline spots, (namely, 1 and 2) were excised from the gel, digested with trypsin, and subjected to reversedphase liquid chromatography separation of peptides with direct sample deposition on a multispot MALDI target. (27) Berkelman, T.; Stenstedt, T. In 2-D Electrophoresis using Immobilized pH Gradients: Principles and Methods; Amersham Pharmacia Biotech: Piscataway, NJ, 1998; pp 17-46. (28) Laemmli, U. K. Nature 1970, 227, 680-685. (29) Cooper, C. A.; Gasteiger, E.; Packer, N. H. Proteomics 2001, 1, 340-349.
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Figure 1. 2D gel electrophoresis of proteins from tomato leaf organellar-free cellular fluids. Proteins (200 µg) from organellar-free cellular fluids were separated by IEF/SDS 2D PAGE and stained with Coomassie Blue R250. Denaturing IEF was carried out using a linear IPG strip of pH 4-7. The numbers above the gel image indicate pI values, and the numbered arrows indicate protein spots analyzed by mass spectrometry.
MALDI MS generally produces singly charged ions [M + H]+ of peptides and glycopeptides, which makes interpretation of the tandem spectra more straightforward than after electrospray ionization MS analysis. Coupling of MALDI MS with HPLC separation greatly increases the resolution and sensitivity for sample analysis by decreasing the number of ions per MALDI spot and therefore decreasing the ion suppression effect. The sequence coverage for the mature protein was 97% for both spots (Figure 1). The protein was present in its mature form; no signal peptide or amino-terminal propeptide could be detected. Glycopeptide-containing fractions were apparent by the presence of glycoform peaks corresponding to the same peptide grouped at higher m/z range (Figure 2, Table 1). Characteristically, the glycopeptide peaks in each fraction spectrum were spaced by m/z 203 (N-acetylglucosamine, GlcNAc), 162 (mannose, Man), or 146 (fucose, Fuc). The relative intensities of ion peaks within each group of glycoforms that were observed for the same glycosylation site showed that the forms with glycan-type paucimannosidic and composition (GlcNAc)2(Man)3(Fuc)1(Xyl)1 could be the most abundant for this protein (total added masses of glycans m/z 1170; corresponding glycopeptide ions m/z 2325.9, 2806.2, 2325.9, and 3784.8; Figure 2). The glycoform with m/z 3476.9 (Table 1) was only found in spot 1 from 2D gels but not in spot 2 (Figure 1). Identification of Glycosylation Sites and Oligosaccharide Composition Analysis. MALDI-QqTOF MS/MS analysis of glycopeptide ions showed a consistent fragmentation pattern (Figure 3) with losses of carbohydrate residues at higher m/z values and the presence of a dominant group of four peaks near the mass of the nonglycosylated peptide itself, namely, [peptide + H - 17]+, [peptide + H]+, [peptide + GlcNAc + H]+ (or [peptide + 204]+), and [peptide + CHCHNHAc]+ (or [peptide + 84]+), the latter corresponding to a 0.2X0 cross-ring fragmentation of the connecting GclNAc residue.25,30 In Figure 3, representative examples of MS/MS spectra are given with one of the glycoforms for each peptide sequence. In all four spectra, the [peptide + H]+ (m/z 1155.5, 1635.8, 2598.3, 1545.7) and [peptide + GlcNAc + (30) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.
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Figure 2. Spectral portions of MALDI single MS analysis containing glycopeptide ion peaks. Characteristic groups of glycopeptide ion peaks spaced by 203 (GlcNAc), 162 (Man) or 146 u (Fuc) were observed in HPLC fractions. (A) Glycosylated forms of GVCESNFTNK detected in fraction 13 from HPLC. (B) Glycosylated forms of TTNSSNPEVIEGFLK detected in HPLC fraction 21. (C) Glycosylation profile of peptide ILAYMNSTSSPVATIAFQGTIIGDK from fraction 23. (D) Glycoforms of ISNATFFTLFDAAK in fraction 25.
H]+ (m/z 1358.6, 1838.9, 2801.4, 1748.8) are the most abundant, along with [peptide + 84]+ (m/z 1238.5, 1718.8, 2681.3, 1628.7) and [peptide + H - 17]+ (m/z 1138.5, 1618.8, 2581.3, 1528.7) ion species. Interestingly, the peptide fragmentation portion of the spectra did not vary significantly with the size of the glycan attached to the peptide. At m/z values lower than this quadruplet, specific y- and b-ions provided most of the peptide sequence information. In fact, the parts of the MS/MS spectra below the m/z value of the [peptide + H]+ were used for automatic interpretation and assignment of peptide identity, using sequencing algorithms of two different independent search engines, Mascot and Sonar MS/MS (Table 1). The search results demonstrated that for most glycopeptides there was sufficient information in the peptide fragmentation pattern to allow the identification of glycoproteins with high confidence. Some peaks, however, were not recognized by the search engines and these corresponded to [y + GlcNAc] and [b + GlcNAc] fragments (y and/or b + 203). These fragment ions are diagnostic signatures for unambiguous location and identification of glycosylation sites in glycopeptides. In addition, [y + 83] and [b + 83] fragments, where 83 corresponds to CHCHNHacetyl, were identified manually as previously described.25 For some glycoforms in Table 1, scores for the search engines’
Table 1. Detection and Identification of Glycopeptides by MALDI-Qq-TOF MS/MS Analysis peptide sequencea
peptideb
GVCESNFTNK
1155.5
TTNSSNPEVIEGFLK
1635.8
ILAYMNSTSSPVATIAFQGTIIGDK ILAYMoxNSTSSPVATIAFQGTIIGDK
2598.3 2614.3
ILAYM-48NSTSSPVATIAFQGTIIGDK
2550.4
ISNATFFTLFDAAK
1545.8
glycopeptideb 2179.9 2325.9 2529.0 2586.0 2732.1 2644.2 2660.0 2806.2 3009.1 3212.2 3622.7 3768.8 3476.9 3638.8 3784.8 3987.7 4190.5 3574.7 3679.5 3720.8 3825.7 3923.9 4028.8 4126.8 4231.7 2570.0 2716.0 2773.1 2919.1 3122.2
Mascot scorec
Sonar scored 10-4
4.3 × 6.3 × 10-4 6.0 × 10-5
27 34 31 17 39 54 59 65 47 49 89 97 88 85 96 88 65
1.4 × 10-6 7.7 × 10-7 6.0 × 10-7 3.8 × 10-7 1.6 × 10-6 3.0 × 10-4 5.6 × 10-13 2.0 × 10-10 3.0 × 10-10 6.0 × 10-15 1.0 × 10-15 4.6 × 10-17 3.1 × 10-15
18 95 56 29 34
1.2 × 10-2 4.3 × 10-8 2.5 × 10-7 4.6 × 10-4 3.5 × 10-5
66 70 61 58 63
4.5 × 10-10 6.3 × 10-10 1.6 × 10-8 3.0 × 10-8 1.3 × 10-7
glycan composition
type
(GlcNAc)2(Man)3(Xyl)1 (GlcNAc)2(Man)3(Fuc)1(Xyl)1 (GlcNAc)3(Man)3(Fuc)1(Xyl)1 (GlcNAc)4(Man)3(Xyl)1 (GlcNAc)4(Man)3(Fuc)1(Xyl)1 (GlcNAc)2(Man)2(Fuc)1(Xyl)1 (GlcNAc)2(Man)3(Xyl)1 (GlcNAc)2(Man)3(Fuc)1(Xyl)1 (GlcNAc)3(Man)3(Fuc)1(Xyl)1 (GlcNAc)4(Man)3(Fuc)1(Xyl)1 (GlcNAc)2(Man)3(Xyl)1 (GlcNAc)2(Man)3(Fuc)1(Xyl)1 (GlcNAc)2(Man)2(Xyl)1 (GlcNAc)2(Man)3(Xyl)1 (GlcNAc)2(Man)3(Fuc)1(Xyl)1 (GlcNAc)3(Man)3(Fuc)1(Xyl)1 (GlcNAc)4(Man)3(Fuc)1(Xyl)1 (GlcNAc)2(Man)3(Xyl)1 (GlcNAc)2(Man)3(Xyl)1 + 105 (GlcNAc)3(Man)3(Fuc)1(Xyl)1 (GlcNAc)2(Man)3(Fuc)1(Xyl)1 + 105 (GlcNAc)3(Man)3(Fuc)1(Xyl)1 (GlcNAc)3(Man)3(Fuc)1(Xyl)1 + 105 (GlcNAc)4(Man)3(Fuc)1(Xyl)1 (GlcNAc)4(Man)3(Fuc)1(Xyl)1 + 105 (GlcNAc)2(Man)3(Xyl)1 (GlcNAc)2(Man)3(Fuc)1(Xyl)1 (GlcNAc)3(Man)3(Xyl)1 (GlcNAc)3(Man)3(Fuc)1(Xyl)1 (GlcNAc)4(Man)3(Fuc)1(Xyl)1
paucimannosidic paucimannosidic complex complex complex paucimannosidic paucimannosidic paucimannosidic complex complex paucimannosidic paucimannosidic paucimannosidic paucimannosidic paucimannosidic complex complex paucimannosidic paucimannosidic paucimannosidic paucimannosidic complex complex complex complex paucimannosidic paucimannosidic complex complex complex
a Sequence tags were identified by HPLC-MALDI-Qq-TOF MS and MS/MS analysis (Figures 1-3) followed by searching MS/MS data sets against nrNCBI database using either MASCOT (Matrix Science Ltd.) or Sonar MS/MS (Proteometrics Ltd.) search engines at