Method for Investigation of Oligosaccharides from Glycopeptides

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Anal. Chem. 2006, 78, 2977-2984

Method for Investigation of Oligosaccharides from Glycopeptides: Direct Determination of Glycosylation Sites in Proteins Erika Lattova´,*,†,‡ Petra Kapkova´,† Oleg Krokhin,§ and He´le`ne Perreault*,†

Chemistry Department and Physics and Astronomy Department, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2, and The Institute of Chemistry, Slovak Academy of Sciences, 842 38 Bratislava, Slovakia

Characterization of glycopeptides has become an important tool toward a better understanding of the molecular details in carbohydrate-protein interactions. In this approach, oligosaccharides are commonly not detectable under mass spectrometric conditions because of ionization suppression by deglycosylated peptides. Their composition is only deduced from the mass differences between glycopeptides and corresponding deglycosylated peptides. Here, we describe how carbohydrates can be easily detected in the PNGase-treated samples and structurally investigated next to the peptides. The efficacy of this method is demonstrated through the analysis of tryptic glycopeptides obtained from human IgG. Following deglycosylation with PNGaseF and derivatization with phenylhydrazine, MALDI spectra produced ion peaks of labeled oligosaccharides and deglycosylated peptides. The relative abundances of individual oligosaccharides were consistent with those of the glycopeptides. MALDI-MS/ MS provided useful data for the structural elucidation of oligosaccharides, including the assignment of dominant isomers and glycosylation sites in peptides. MALDI-MS/ MS fragmentation patterns of deglycosylated peptide ions indicated glycosylation sites at asparagine 297 and 299. The observed peptide of the composition ADQTVYR, described for the first time in this study, indicated new glycosylation sites in IgG1 human myeloma plasma. Glycosylation is one of the most common forms of posttranslational modifications in proteins. Oligosaccharides linked to specific sites can play many various roles in the structure and function of proteins and in the spacing and orientation of cell surface proteins.1,2 Therefore, the detailed understanding of glycoprotein structure also has an impact on medical applications such as the development and the delivery of new vaccines formulated to provoke an antibody response within a selected isoform.3 * Corresponding authors. E-mail: [email protected]; perreau@ cc.umanitoba.ca. Phone: 204-474-6561. Fax: 204-474-7608. † Chemistry Department, University of Manitoba. ‡ Slovak Academy of Sciences. § Physics and Astronomy Department, University of Manitoba. (1) Helenius, A.; Aebi. Science 2001, 291, 2364. (2) Rudd, P. H.; Guile, G. R.; Ku ¨ ster, B.; Harvey, D. J.; Opdenakker, G.; Dwek, R. A. Nature 1977, 388, 205-207. 10.1021/ac0519918 CCC: $33.50 Published on Web 03/30/2006

© 2006 American Chemical Society

The most traditional methods for structural investigation of protein-bound oligosaccharides employ enzymatic or chemical release of the carbohydrate moieties from the intact glycoprotein.4-6 The released glycans can then be analyzedsmonitoring by means of gel filtration,7 HPLC,8,9 or mass spectrometric methods.10 However, the analysis of carbohydrates obtained from the intact glycoproteins has a serious limitation. Because glycans are cleaved nonspecifically, the determination of glycosylation sites cannot be achieved.11 Over the past decade, MALDI-MS analyses of tryptic glycopeptides obtained by gel-trypsin digestion have been reported.12 Glycopeptides could only be detected after removal of the terminal sialic residues if the peptide mixture was not first separated by reversed-phase HPLC. A drawback to this approach is that glycopeptides may not be detected among several other nonglycosylated peptides, also present in the digest. These tend to suppress the signals of generally less abundant glycopeptides. Using Pronase instead of trypsin, for digestion, nonglycosylated peptides of ovalbumin and ribonuclease B were shown susceptible to complete digestion. Mass spectra showed mostly glycopeptide moieties with masses over 1000 Da.13 In the above-mentioned methods, isolated glycopeptides are subsequently treated with a glycosidase to cleave the oligosaccharide. The difference in mass, following a sugar detachment, is used to infer the carbohydrate constituents. Generally, these methods do not allow detection of oligosaccharides, determination of their structure, or the exact site of their attachment to the peptides.14 (3) Jefferis, R.; Lund, J.; Pound, J. D. Immunol. Rev. 1998, 163, 59-76. (4) Tarentino, A. L.; Gomez, C. M.; Plummer, T. H. Biochemistry 1985, 24, 4665-4671. (5) Takasaki, S.; Mizuochi, T.; Kobata, A. Methods Enzymol. 1982, 83, 263268. (6) Patel, P.; Bruce, J.; Merry, A.; Bigge, C.; Wormald, M.; Jaques, A.; Parekh, R. Biochemistry 1993, 32, 679-693. (7) Dwek, R. A.; Edge, C. J.; Harvey, D. J.; Wormald, M. R.; Parekh, R. B. Annu. Rev. Biochem. 1993, 62, 65-100. (8) Rudd, P. M.; Dwek, R. A. Curr. Opin. Biotechnol. 1997, 8, 488-497. (9) Rudd, P. M.; Colominas, C.; Royle, L.; Murphy, N.; Hart, N.; Merry, H. A.; Hebestreit, H. F.; Dwek, R. A. Proteomics 2001, 1, 285-294. (10) Harvey, D. J. Proteomics 2005, 5, 1774-1786. (11) Ku ¨ ster, B.; Wheeler, S. F.; Hunter A. P.; Dwek, R. A.; Harvey, D. J. Anal. Biochem. 1997, 250, 82-101. (12) Mortz, E.; Sareneva, T.; Haebel, S., Julkunen, I.; Roepstorff, P. Electrophoresis 1996, 17, 925-931. (13) An, H. J.; Peavy, T. R.; Hedrick, J. L.; Lebrilla, C. B. Anal. Chem. 2003, 75, 5628-5637. (14) Choudhary, G.; Schwartz, J.; Cho, D. Pharm. Discovery 2005, May 1.

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This article demonstrates how oligosaccharides detached from glycopeptides can be easily visualized under MALDI-MS conditions and structurally investigated next to their peptide. As a model glycoprotein for this study, commercial immunoglubulin IgG from human plasma was chosen. IgG is a multifunctional glycoprotein which basic structure consists of two heavy and two light chains in covalent and noncovalent associations that form three independent protein moieties connected through a flexible linker. The conserved asparagine glycosylation site (Asn 297) is located in the CH2 domains.15 The presence of oligosaccharides is essential for the expression of IgG functions and has a variable influence on the efficacy of specific functions. For mass spectrometric analysis, carbohydrates are most often converted into derivatives, which can simplify spectral interpretation. Several derivatization techniques have been described to produce labeled derivatives of carbohydrates by reductive amination.17-22 Here, for detection of carbohydrates cleaved from glycopeptides, nonreductive phenylhydrazine (PHN) tagging was chosen because this method allows the direct analysis of mixtures, i.e., without sample cleanup.23,24 The derivatization conditions used for PHN tagging enabled simultaneous investigation of detached oligosaccharides next to the peptides, both compounds originating from “mother glycopeptides”. Tagged oligosaccharides and deglycosylated peptides can be analyzed at once, without the necessity of the separation prior to the sugar analysis. METHODS Materials and Reagents. Polyclonal IgG (human plasma) and IgG1 (human myeloma plasma) were purchased from Fitzgerald (Concord MA). PHN and 2,5-dihydroxybenzoic acid (DHB) were obtained from Sigma (St. Louis, MO). Peptide-N-glycosidase F (PNGaseF) deglycosylation kits were purchased from Prozyme (San Leandro, CA). Solvents (acetonitrile, ethanol) were HPLCgrade and obtained from Fisher Scientific (Fair Lawn, NJ). HPLCgrade water was obtained with a Milli-Q plus TOC water purification system (Millipore, Bedford, MA). Trypsin Digestion. Intact glycoprotein (0.5 mg) was dissolved in 25 mM ammonium bicarbonate (200 µL) and digested with trypsin (5 µg) at 37 °C for 20 h. After the incubation, the digest was frozen to terminate the reaction and then evaporated to volume, ∼20 µL. Half of this volume was used for one HPLC separation. RP-HPLC Fractionation of the Tryptic Digest. The tryptic glycopeptides were fractionated and purified on a System Gold HPLC chromatograph equipped with a System Gold 166 UV detector and 32-Karat software (Beckman-Coulter, ). For reversedphase HPLC, the analytical column Vydac 218 TP54 Protein (15) Parekh, R. B.; Dwek, A.; Sutton, J.; et al. Nature 1985, 316, 452-457. (16) Raju, T. S.; Briggs, J. B.; Borge, S. M.; Jones, A. J. S. Glycobiology 2000, 10, 477-486. (17) Hase, S.; Ibuki, T.; Ikenaka, T. J. Biochem. (Tokyo) 1984, 95, 197-203. (18) Strydom, D. J. J. Chromatogr., A 1994, 678, 17-23. (19) Bigge, J.; Patel, T. P.; Bruce, J. A.; Goulding, P. N.; Charles, S. M.; Parekh, R. B. Anal. Biochem. 1995, 230, 229-238. (20) Suzuki, S.; Kakehi, K.; Honda, S. Anal. Chem. 1996, 68, 2073-2083. (21) Camilleri, P.; Tolson, D.; Birrell, H. Rapid Commun. Mass Spectrom. 1998, 12, 144-148. (22) Franz, A. H.; Molinski, T. F.; Lebrilla, C. B. J. Am. Soc. Mass Spectrom. 2001, 12, 1254-1261. (23) Lattova, E.; Perreault, H. J. Chromatogr., B 2003, 793, 167-179. (24) Lattova, E.; Snovida, S.; Krokhin, O.; Perreault, H. J. Am. Soc. Mass Spectrom. 2005, 16, 683-696.

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&Peptide C18 (Separation Group, Hesperia, CA) was used. The chromatograph was equipped with a Rheodyne injector (5-µL loop). The samples (10 µL injected) were eluted with 5% ACN in water as solvent A and 90% ACN in 0.1% TFA as solvent B at a flow rate 1 mL/min. An elution gradient was applied from 5 to 65% ACN over 90 min. UV detection was performed at 245 nm. All fractions were collected manually, including those with peaks of very low intensities in UV-HPLC chromatogram and concentrated in vacuo prior to MS analysis. Deglycosylation of Tryptic Glycopeptides with PNGaseF. Selected fractions of glycopeptides in water (10 µL) and incubation buffer (2.5 µL) were incubated with PNGaseF (0.3 µL) for 18 h at 37 °C,25 without using denaturating detergent. Derivatization of Oligosaccharide. PNGaseF-treated fractions of glycopeptides (∼30 µL of water) were derivatized with phenylhydrazine (0.3 µL) at 70 °C for 1 h (periodically mixed during incubation). In the case of on-target derivatization, a phenylhydrazine solution (0.5 µL; 2 µL of PHN in 10 µL of water) and a deglycosylated solution (∼1 µL) were spotted together onto dried DHB on the target and allowed to react at 37 °C (1-2 h). MALDI Mass Spectrometric Analysis. MALDI mass spectra were recorded on a Biflex-IV spectrometer (Bruker Daltonics, Billerica, MA). The accelerating voltage was 20 kV. Profiling of the molecular ions in the samples was performed in positive and negative ion polarities, using reflective and linear time-of-flight (TOF) modes. MALDI-MS/MS spectra were acquired using a prototype quadrupole-quadrupole-TOF (QqTOF) mass spectrometer with photon pulses from a 20-Hz nitrogen laser (VCL 337ND, SpectraPhysics, Mountain View, CA) with 300 mJ energy/pulse.26 The collisional energy for each precursor ion was determined by applying a well-defined accelerating voltage at the entrance of the collisional cell, and values were ∼50 eV/1000 Da. The sample solution (∼0.5 µL) was loaded onto a stainless steel target with the same volume of the saturated matrix solution (DHB in acetonitrile-water 1:1) and allowed to air-dry. RESULTS AND DISCUSSION Generally, oligosaccharides and peptides from glycoproteins are analyzed separately. In ideal cases, it should be convenient to detect both groups from one MALDI-target spot, especially when limited amounts of biological material are available. In a first set of experiments, a mixture of four common peptide standards (angiotensins I and II, bombesin, and ACTH) was added to two oligosaccharide standards (molar ratio of peptide to oligosaccharide 1:2) and subjected to MALDI-MS. Only abundant peptide ions were detected in the spectrum (Figure 1a). The peptide-oligosaccharide mixture was then reacted with PHN to help visualize the peaks corresponding to oligosaccharide standardssthe sugar ion peaks were approximately as high as those ion peaks corresponding to the peptides. After 2 h of heating, the peptide peaks had nearly disappeared, leaving abundant oligosaccharides ions in the spectrum (Figure 1b).27 Derivatization in this case provides better detection of oligosaccharides, and (25) Tarentino, A. L.; Plummer, T. H. Methods Enzymol. 1994, 230, 44-57. (26) Loboda, A. V.; Krutchinsky, N. N.; Bromirski, M. P.; Ens, W.; Standing, K. G. Rapid Commun. Mass Spectrom. 2000, 14, 1047-1057. (27) Lattova, E.; Krokhin, O.; Wilkins, J. A.; Perreault, H. 52nd ASMS Nashville, TN, 2004; ThPB 028.

Figure 1. Positive MALDI-MS spectra of an artificially prepared mixture (1:2) of peptides (angiotensins II and I, bombesin, ACTH)) and oligosaccharide standards; * (NGA2F, NA2F): (a) before reaction; (b) after 2 h of heating with PHN at 80 °C.

depending on the reaction conditions, peptides can also be analyzed. This phenomenon has been useful for direct MS detection of oligosaccharides detached from glycopeptidesswhich otherwise are not detectable because of suppression by deglycosylated peptide ions. For the purposes of this study, IgG was first treated with trypsin and the digest was fractionated on the reversed-phase HPLC; all fractions were collected manually and analyzed with MALDI-TOF MS. The peaks with mass differences of 162 and 203 Da indicated the presence of hexose and N-acetylhexosamine residues, respectively. Sialylated glycopeptides were identified according to 291 Da differences and fucosylated glycopeptides according to 146 Da mass shifts. Isolated glycopeptide fractions were enzymatically deglycosylated with PNGaseF. In the MALDIMS spectra of deglycosylated samples, again, only peptide peaks were identified. Subsequent derivatization of these mixtures with PHN (on-target or in-tube) helped to observe cleaved oligosaccharides. The masses of detached glycans were compared with carbohydrate structures found in a previous study on glycosylation of human IgG16 and confirmed by MALDI-MS/MS. It is important to point out that the total identification of glycoprotein IgG was not the primary intention of this study. Our main goal was to demonstrate the capability and performance of the method for direct identification of carbohydrates detached from glycopeptides. IgG Polyclonal Human Plasma. Glycopeptides first eluted from the RP column (m/z 3087-3306) indicated the presence of sialic acid (Table 1, FR.1). Derivatization of fraction 1 after deglycosylation gave rise to sialylated oligosaccharides that were observed in the negative MALDI-MS spectrum, coherently with

[M - H]- peptide ions at m/z 1189.6 (Figure 2). The highest peak at m/z 2166.6 could be as assigned to an oligosaccharide of the composition Gal2(GlcNAc)4Man3FucSA and was detached from glycopeptide detected at m/z 3249.9 (Figure 2a). The peak at m/z 2004.9 was consistent with a structure containing one hexose residue less than the previous one (162 u less) and associated with the glycopeptide detected at m/z 3087.6. The sialylated oligosaccharides observed with very low abundances at m/z 2207.5, 2224.3, and 2370.1 originated from glycopeptides at m/z 3290.7, 3306.7, and 3452.6, which were hardly detectable by MS (Figure 2). These glycans corresponded to sialylated structures with bisecting GlcNAc moieties. The second HPLC fraction (Table 1, FR.2) comprised the glycopeptides with m/z observed at 2431-3162 (Figure 3a). After deglycosylation, these compounds produced [M + H]+ peptide ions at m/z 1190.5 of same composition (vide infra) as observed in the fraction shown above (Figure 3b). On-target derivatization of this spot helped to detect peaks that were consistent with masses of labeled neutral oligosaccharides (Figure 3c,d). The large peak at m/z 2634.7 in Figure 3a corresponded to a glycopeptide with an oligosaccharide component (GlcNAc)4Man3Fuc. The glycopeptide detected at m/z 2796.8 contained an additional hexose residuesGal(GlcNAc)4Man3Fuc. A less abundant glycopeptide observed at m/z 2837.8 included a carbohydrate moiety of (GlcNAc)5Man3Fuc composition. Ions at m/z 2958.6 corresponded to a glycoform with composition Gal2(GlcNAc)4Man4Fuc. The peak shifted by 41 u (m/z 2999.7) contained a GlcNAc residue substituted for hexose. The small peak at m/z 3161.9, obtained after derivatization, was related to the oligosaccharide peak at m/z 2103.2 assigned to a fucosylated structure Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

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Table 1. Peak Assignments for MALDI-MS Measurements of Glycopeptides, Derivatized Glycans, and Deglycosylated Peptides Found in IgG Polyclonal Human Plasma observed m/z values

glycopeptide [M + H]+

oligosaccharide (as PHN) [M + Na]+ or *[M - H]- a

peptide (deglycosylated) [M + H]+ or *[M - H]-

FR.1

3087.6 3103.8 3249.9 3290.7 3306.7 3452.6

2004.9* (H4N4FS) 2021.9* (H5N4S) 2166.6* (H5N4FS) 2207.5* (H4N5FS) 2224.3* (H5N5S) 2370.1* (H5N5FS)

1189.47* (obs) 1189.497* (calc) 295EEQYDSTYR303

FR.2

2431.3 2488.4 2634.7 2650.5 2691.7 2796.8 2812.6 2837.8 2958.6 2999.7 3161.9

1372.8 (H3N3F) 1429.7 (H3N4) 1575.9 (H3N4F) 1591.9 (H4N4F) 1632.8 (H3N5) 1737.8 (H4N4F) 1753.9 (H5N4) 1778.9 (H3N5F) 1900.1 (H5N4F) 1941.1 (H4N5F) 2103.2 (H5N5F)

1190.503 (obs) 1190.497 (calc. 295EEQYDSTYR303

FR.3

2618.9 2781.1 2943.4 2984.5

1575.8 (H3N4F) 1737.9 (H4N4F) 1899.8 (H5N4F) 1941.1 (H5N4F)

1174.509 (obs) 1174.502 (calc) 293EEQFDSTYR303

FR.4

2456.2 2561.3 2602.3 2764.4 2805.4 2926.7 2967.8 3129.7

1429.6 (H3N4 1534.7 (H4N3F) 1575.4 (H3N4F) 1738.0 (H4N4F) 1778.8 (H3N5F) 1900.3 (H5N4F) 1940.9 (H4N5F) 2103.1 (H5N5F)

1158.498 (obs) 1158.507 (calc) 293EEQFDSTYR301

Figure 2. (a) Positive MALDI-MS spectrum acquired from HPLC Fraction 1 (el. time 4 min) after trypsin digestion of polyclonal IgG whole, (*) nonglycosylated peptide. (b) Negative MALDI-MS spectrum of fraction 1 acquired after enzymatic deglycosylation with PNGaseF and derivatization with PHN. All ions are as [M - H]-. Symbols: 2 Fuc; b Gal; O Man; 9 GlcNAc; [ NeuAc.

a Key: H ) hexose (Man, Gal); N ) GlcNAc; F ) fucose; S ) sialic acid (NeuAc).

with two galactosyl residues and a bisecting GlcNAc moietys Gal2(GlcNAc)5Man3Fuc. The other small glycopeptide peaks at m/z 2431.3 and 2488.4 in Figure 3a corresponded to very small peaks at m/z 1372.8 and 1429.7 in Figure 3c,d, i.e., glycans of (GlcNAc)4Man3 and (GlcNAc)3Man3Fuc compositions. Deglycosylation of glycopeptides found in fraction 3 (Table 1, FR.3) produced [M + H]+ peptide ions at m/z 1174.5. After derivatization, observed peaks were consistent with the masses of carbohydrates as described above. Figure 4 presents MALDI-MS spectra acquired from the last fraction (Table 1, FR.4). Glycopeptides detected at m/z 24563130 provided a peptide with [M + H]+ ions observed at m/z 1158.5. The peaks of derivatized oligosaccharides were in accordance with those observed in fractions 2 and 3. IgG1 Human Myeloma Plasma. Glycopeptides analyzed from the first HPLC fraction (m/z 2634-3405) indicated the presence of neutral and sialylated oligosaccharides (Figure 5). After enzymatic and derivatization treatment, two deglycosylated peptides were detected with [M + H]+ ions at m/z 852.4 and 1190.5. The peptide with higher m/z in this fraction was mostly related to masses of neutral glycoforms. The peptide at m/z 852.4 was associated mainly with sialylated species (Figure 5c; see Table 2, FR.1). 2980 Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

Figure 3. Positive MALDI-MS spectra acquired from HPLC fraction 2 (el. time 8 min) from polyclonal IgG. (a) After trypsin digestion, all ions are [M + H]+; (b) after enzymatic deglycosylation with PNGaseF; (c) on-target derivatized with PHN, oligosaccharide ions are [M + Na]+; (d) shows an expansion of the spectrum in (c), recorded from m/z 1300.

In the second fraction, two large peaks indicated carbohydrate constituentssm/z 2661.5 and 2823.9 (Figure 6a). After enzymatic release of oligosaccharides, the deglycosylated peptide was observed at m/z 852.4 and on-target derivatization yielded peaks at m/z 1940.7 and 2102.8, which were consistent with masses of labeled neutral oligosaccharides (Figure 6b). MALDI-MS/MS of Cleaved Oligosaccharides. MALDI-MS/ MS spectra acquired for detached derivatized glycans showed

Table 2. Peak Assignments for MALDI-MS Measurements of Glycopeptides, Derivatized Glycans, and Deglycosylated Peptides Found in IgG Polyclonal Human Plasma observed m/z values

glycopeptide [M + H]+

oligosaccharide (as PHN) [M + Na]+ or *[M - H + 2Na]+ or **[M - 2H + 3Na]+ a

2912.4 2952.2 3114.2 3405.3

2213.8* (H4N4FS) 2253.8* (H5N4FS) 2415.8* (H5N5FS) 2729.0** (H2N5FS2)

852.422 (obs) 852.424 (calc) ADQTVYR

FR.1

2634.2 2650.1 2796.3 2812.2 2837.4 2958.2 2999.9 3016.0 3161.9 3249.5

1575.6* (H3N4S) 1591.5* (H4N4) 1737.6 (H4N4F) 1753.6 (H5N4) 1778.9 (H3N5F) 1899.9 (H5N4F) 1940.8 (H4N5F) 1956.9 (H5N5) 2103.2 (H5N5F) 2213.8* (H5N4FS)

1190.503 (obs) 1190.497 (calc) 295EEQYDSTYR303

FR.3

2661.5 2823.9

1940.7 (H4N5F) 2102.8 (H4N5F)

852.422 (obs) 852.424 (calc) ADQTVYR

Figure 4. Positive MALDI-MS spectra acquired from HPLC fraction 4 (el. time 28 min) from polyclonal IgG whole. (a) After trypsin digestion, all ions are [M + H]+; (b) after enzymatic deglycosylation with PNGaseF and on-target derivatization with PHN, oligosaccharide ions are [M + Na]+.

peptide (deglycosylated) [M + H]+

a Key: H ) hexose (Man, Gal); N ) GlcNAc; F ) fucose; S ) sialic acid (NeuAc).

Figure 5. Positive MALDI-MS spectra acquired from glycopeptides of HPLC fraction 1 (el. time 4 min) from IgG1. (a) After trypsin digestion, all ions are [M + H]+; (b) after enzymatic deglycosylation with PNGaseF and on-target derivatization with PHN, [M + Na]+ oligosaccharide ions; (c) shows an expansion of the spectrum in (b), recorded from m/z 1500.

oligosaccharide structures. Some of these spectra will be discussed next. The oligosaccharide observed at m/z 1575.6 ([M + Na]+) provided a fragmentation pattern supporting a structure with GlcNAc on each arm and Fuc linked at the 6-position of the terminal labeled GlcNAc (2,4A5 at m/z 1178.6 and B3/Y3 at m/z 550.2). The MALDI-MS/MS spectrum of m/z 1737.5 precursor ions (Figure 7a) confirmed a structure with 6-substitution of the fucose on the reducing GlcNAc residue (cross-ring ions 4,5A6 at m/z 1546.5 and 2,4A6 at m/z 1340.4). The loss of hexosyl from B4 fragments produced abundant ions at m/z 915.3, consistent with isomer 1sgalactose linked at GlcNAc of 3-positioned mannose and supported by cross-ring cleavage ions at m/z 1181.4 (2,3X3).

Figure 6. Positive MALDI-MS spectra acquired from HPLC fraction 2 glycopeptides (el. time 28 min) from IgG1. (a) After trypsin digestion; (b) after enzymatic deglycosylation with PNGaseF and derivatization with PHN, [M + Na]+ oligosaccharide ions.

Less abundant B4/Y4β ions at m/z 874.3 (loss of GlcNAc) were associated with isomer 2 in which galactose is linked at the 6-arm. According to MS/MS results, isomer 1 with galactose substitution on the 3-arm was dominant not only in the IgG1 sample but also in whole IgG. This is in agreement with experimental results, which indicate that a biantennary oligosaccharide in free solution is galactosylated preferentially on the ManR(1f3) arm, relative to the ManR(1f6) arm.28 Fragmentation patterns observed for Gal(GlcNAc)5Man3FucPHN glycans ([M + Na]+ at m/z 1940.7) were supportive of (28) Fuji, S.; Nishiura T.; Nishikawa A.; Miura, R.; Taniguchi N. J. Biol. Chem. 1990, 265, 6009-6018.

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Figure 7. MALDI-MS/MS spectra of galactosylated glycans detected. (a) At m/z 1737.5 in HPLC fraction 1 (IgG1 myeloma plasma); (b) at m/z 1940.7 from HPLC fraction 3 (IgG1myeloma plasma); (c) at m/z 2102.8 from HPLC fraction 3 (IgG1 myeloma plasma); (d) at m/z 2415.9 (fraction 1 of IgG1 myeloma plasma); (e) at m/z 2728.9 (fraction 1 of IgG1 myeloma plasma). Fragment ions are sodiated.

the two possible structures, 1 and 2 (Figure 7b). Isomer 1 produced B4/Y5R fragment ions (loss of Gal residue) at m/z 1118.4, and prominent ions at m/z 532.2 (loss of 221 u from B4/Y3R) arose from the loss of bisecting GlcNAc. Structure 2 was supported by the loss of GlcNAc from the 3-antenna (B4/Y4β at m/z 1077.4). Fragment ions at m/z 915.3 (B4/Y3β) and the associated loss of 221 u (bisecting GlcNAc) also indicated Gal substitution on the 6-antenna.29 Fragment ions in the MALDI-MS/MS spectrum of m/z 2102.8 ions (composition Gal2(GlcNAc)5Man3FucPHN) indicated a structure bearing galactose residues on both arms (Figure 7c). The cleavage ions at m/z 915.3 (B4/Y3R) followed by an abundant loss 2982 Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

of bisecting GlcNAc (m/z 694.2) were consistent with the presence of a second galactose linked to GlcNAc on the 6-arm. Glycans of composition Gal2(GlcNAc)5Man3FucSAPHN, with parent ions at m/z 2415.9 ([M - H + 2Na]+) produced the fragmentation pattern shown in Figure 7d. Cross-ring cleavage ions (2,4A7 at m/z 2018.7 and 1,6A6 at m/z 1911.7) were supportive for the linkage of fucose at the 6-position of the labeled reducing GlcNAc residue. Ions containing sialic acidsB6 at m/z 1958.6 and C5 at m/z 1771.6swere consistent with the loss of the chitobiose (29) Lattova, E.; Krokhin, O.; Perreault, H. J. Am. Soc. Mass Spectrom. 2004, 15, 725-735.

Figure 8. MALDI-MS/MS spectra of deglycosylated peptides detected at m/z (a) 1190.5, (b) 1174.5, (c) 1158.5, and (d) 852.4.

core. The presence of bisecting moiety appeared as abundant ions at m/z 694.2 (loss of 221 mu). The largest oligosaccharide observed (m/z 2728.9, [M 2H + 3Na]+) occurred in very low abundance and its MALDIMS/MS spectrum was noisier than others, however, sufficiently detailed for structural investigation (Figure 7e). The fragmentation pattern corresponded to a disialylated structure of Gal2(GlcNAc)5Man3FucSA2PHN composition. Y6, cleavage of a sodiated sialic acid residue (313 u) produced a peak at m/z 2415.9, and the loss of a second sodiated sialic moiety yielded m/z 2102.8 ions ((Y6)2). Disialylated fragment ions, B6 at m/z 2271.8 and C5 at m/z 2084.6, corresponded to the cleavage of the fucosylated chitobiose core and were supportive of the presence of sialic acids on both arms. The presence of bisecting GlcNAc was evidenced by the loss of 221 u from B5/Y3R ions and produced abundant ions at m/z 1007.6. MALDI-MS/MS of Deglycosylated Peptides. Figure 8 shows MALDI-MS/MS spectra of deglycosylated peptides ob-

served after PNGaseF/PHN-derivatization treatment of isolated glycopeptides. The peptide with m/z at 1190.503 (observed in polyclonal IgG and IgG1myeloma) provided a fragmentation pattern consistent with the following sequence of amino acidssEEQYDSTYR (Figure 8a). The sequence could correspond to a EEQYNSTYR residue 295-303, i.e., with asparagine at position 299, found in human monoclonal IgG as a nonglycosylated peptide (H-chain, IgG1, subgroup III).30 MS/MS analysis of a deglycosylated peptide from polyclonal IgG with [M + H]+ ions at m/z 1174.509 (Figure 8b) confirmed the presence of phenylalanine instead of tyrosine next to asparaginesEEQFDSTYR (sequenced manually). The peptide observed at m/z 1158.498 (polyclonal IgG) was shown to contain two phenylalanines (EEQFDSTFR) according (30) Postingl, H.; Hilschmann, N. Hoppe Seylers Z. Physiol. Chem. 1976, 357, 1571-604.

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to its MS/MS fragmentation pattern (sequenced manually, Figure 8c). Peptides observed at m/z 1174.5 and 1158.5 matched the residues described by Sondermann et al.31 in the Cγ2 domains of hFc1 in human and mouse immunoglobulins, with glycosylation sites at asparagine 297. The peptide that produced parent ions at m/z 852.422 was observed only in the IgG1 from myeloma plasma. The fragmentation pattern (sequenced manually) was associated with the amino acid sequence ADQTVYR (Figure 8d). This sequence did not match with any peptide described for IgG. In one study, a human IgG1 myeloma protein was shown to be glycosylated in the VL region, in addition to IgG-Fc.32 It may be possible that the deglycosylaled peptide of the composition ADQTVYR comes from the light or heavy chain. This issue needs to be explored further, but in the case of this article, it has helped to show the versatility and usefulness of our method. CONCLUSION Derivatization of PNGase-treated glycopeptide samples obtained from tryptic digests of IgG samples enabled the observation of oligosaccharide peaks next to their deglycosylated peptides. The masses of labeled carbohydrates were in good accordance with IgG oligosaccharides found in previous studies. The relative abundances of individual oligosaccharides were consistent with those of glycopeptides. MALDI-MS/MS spectra provided important insights on the determination of dominant isomers. The fragmentation patterns of PHN-oligosaccharides were supportive of galactose dominantly linked on the ManR(1f3) arm. Sialylated (31) Sondermann, P.; Huber, R.; Oosthulzen, V.; Jacob, U. Nature 2000, 406, 267-273. (32) Jefferis, R. Biotechnol. Prog. 2005, 21, 11-16.

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oligosaccharides provided very good fragmentation patterns, as sialylated fragments were produced. After PHN treatment, deglycosylated peptides still produced sufficiently strong signals to allow the acquisition of MS/MS spectra. Sequencing helped to indicate the deglycosylated sites. Two deglycosylated peptides, EEQFDSTYR and EEQFDSTF, have been previously described in the monoclonal human IgG-Fc fragment in Cγ2 domains with glycosylation sites at asparagine 297.31 The deglycosylated peptide observed at m/z 1190.5, with composition EEQYDSTYR, corresponded to the residue 295-303 (glycosylated site at asparagine 299) segment found in the monoclonal IgG1-H-chain.30 The deglycosylated peptide with parent ions at m/z 852.4 yielded a fragmentation pattern indicating a new sequence with composition ADQTVYR. This last residue did not match to any known IgG peptide. Finding the exact location of this peptide would necessitate separate investigations of the light and heavy chains of IgG1. ACKNOWLEDGMENT The authors thank Professor Roy Jefferish from the University of Birmingham (The School of Medicine) for helpful discussion. Acknowledgments are made to Professors Kenneth G. Standing and Werner Ens for the use of the Qq-TOF mass spectrometer. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), from the Canadian Foundation for Innovation (CFI), and the Canada Research Chairs Program (CRC) for funding.

Received for review November 8, 2005. Accepted February 27, 2006. AC0519918