Complete and Rapid Peptide and Glycopeptide Mapping of Mouse

Complete and Rapid Peptide and Glycopeptide Mapping of Mouse Monoclonal Antibody by LC/MS/MS Using Ion Trap Mass Spectrometry. Kazuo Hirayama* ...
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Anal. Chem. 1998, 70, 2718-2725

Complete and Rapid Peptide and Glycopeptide Mapping of Mouse Monoclonal Antibody by LC/MS/MS Using Ion Trap Mass Spectrometry Kazuo Hirayama,*,† Reiko Yuji,† Naoyuki Yamada,† Koichi Kato,‡ Yoji Arata,‡,§ and Ichio Shimada‡

Central Research Laboratories, Ajinomoto Company, Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan, and Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Tokyo 113-0033, Japan

Complete and rapid peptide and glycopeptide mapping of a mouse monoclonal immunoglobulin (IgG2b) were carried out by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry (LC/ ESI IT-MS/MS). It was possible to obtain spectra of a minor glycopeptide at a quantity as low as 1.8 pmol. Reduced and carboxymethylated mouse antidansyl monoclonal IgG2b (RCM-IgG2b) was digested with lysyl-endopeptidase. Proteolytic peptides were subjected to capillary HPLC separation followed by analysis with an ion trap mass spectrometer. The complete amino acid sequence of the IgG2b was characterized by using LC/ ESI IT-MS/MS. The structures of 12 different types of O-linked oligosaccharides attached to Thr-221AH in the hinge region and those of three major types of N-linked oligosaccharides attached to Asn-297H have been characterized. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) have greatly expanded the analytical utility of mass spectrometry (MS) for the structural characterization of biological macromolecules. Determination of both the molecular weight and the sequence of peptides derived by enzymatic digestion of proteins can be achieved by LC-ESI and MALDI-time-of-flight (TOF) mass spectrometry. A great advantage of both LC-ESI and MALDI-TOF mass spectrometry is that only a small amount of sample is required for the determination of the molecular weight and the sequence characterization of peptides derived from macromolecules such as proteins. However, in most cases, structural characterization of proteins by these two types of mass spectrometry has been carried out only by using molecular weight information of the peptides derived by digestion with specific enzymes. Accordingly, some structural ambiguity remains when larger proteins are to be analyzed. In fact, unexpected cleavages sometimes occur in the proteolytic digestion of proteins.1,2 Furthermore, it is difficult to completely characterize the sugar sequences of N- or O-linked oligosaccharides in

glycoproteins without using both MS and MS/MS methods.3,4 Mass spectrometric analyses of the whole molecule and fragments of immunoglobulin G (IgG), which has a molecular mass of ∼150 ku, has been carried out by ESI-MS5-8 or MALDITOFMS.9 In addition, peptide mappings of a recombinant humanized monoclonal antibody have been carried out by using ESIMS10,11 or MALDI-TOFMS11 and liquid secondary ionization mass spectrometry (LSIMS).11 In these studies, it was not possible to detect all molecular masses of digested peptides by LC/ESI-MS or MALDI-TOFMS analyses. Roberts et al.12 reported structural characterization of a recombinant, reshaped human monoclonal antibody by mass spectrometry. It was possible to achieve the structure verification for 99.1% of the light-chain and for 99.3% of the heavy-chain amino acid sequences of the recombinant product, including the structure of the N-linked sugar chain. We have reported the complete characterization by MALDI-TOFMS and LC/ESI-MS of the amino acid sequences of mouse monoclonal antibodies and the molecular weight of digested peptides.13 IgG consists of two identical heavy and light chains. The heavy chain is composed of four homologous units, VH (variable region of the heavy chain), CH1, CH2, and CH3 (constant regions of the heavy chain), whereas the light chain is divided into two homologous units, VL (variable region of the light chain) and CL (constant region of the light chain). An N-linked oligosaccharide chain is attached to Asn-297H in the CH2 domain. It has been

* Reprint requests: (e-mail) [email protected]; (tel) +81-44-244-1498; (fax) +81-44-211-7609. † Ajinomoto Co., Inc. ‡ The University of Tokyo. § Present address: Water Research Institute, Sengen 2-1-6, Tsukuba, Ibaraki 305-0047, Japan. (1) Johnson, R. S.; Biemann, K. Biochemistry 1987, 26, 1209-1214.

(2) Johnson, R. S.; Mathews, W. R.; Biemann, K.; Hopper, S. J. Biol. Chem. 1988, 263, 9589-9597. (3) Huddleston, M. J.; Bean, M. F.; Caa, S. A. Anal. Chem. 1993, 65, 877-884. (4) Rush, R. S.; Derby, P. L.; Smith, D. M.; Merry, C.; Rogers, G.; Rohde, M. F.; Katta, V. Anal. Chem. 1995, 67, 1442-1452. (5) Feng, R.; Konishi, Y. Anal. Chem. 1992, 64, 2090-2095. (6) Feng, R.; Konishi, Y. Anal. Chem. 1993, 65, 645-649. (7) Ashton, D. S.; Beddell, C. R.; Cooper, D. J.; Craig, S. J.; Lines, A. C.; Oliver, R. W. A.; Smith, M. A. Anal. Chem. 1995, 67, 835-842. (8) Bourell, J. H.; Clauser, K. P.; Kelley, R.; Carter, P.; Stults, J. T. Anal. Chem. 1994, 66, 2088-2095. (9) Siegel, M. M.; Hollander, I. J.; Hamann, P. R.; James, J. P.; Hinman, L.; Smith, B. J.; Farnsworth, A. P. H.; Phipps, A.; King, D. J.; Karas, M.; Ingendoh, A.; Hillenkamp, F. Anal. Chem. 1991, 63, 2470-2481. (10) Lewis, D. A.; Guzzetta, A. W., Hancock, W. S., Costello, M. Anal. Chem. 1994, 66, 585-595. (11) Cano, L.; Swiderek, K. M.; Shively, J. E. In Techniques in Protein Chemistry VI; Crabb, J. W., Ed.; Academic Press: San Diego, 1995; pp 21-30. (12) Roberts, G. D.; Johnson W. P.; Burman, S.; Anumula, K. R.; Carr, S. A. Anal. Chem. 1995, 67, 3613-3625. (13) Akashi, S.; Noguchi, K.; Yuji, R.; Tagami, U.; Hirayama, K.; Kato, K.; Kim, H.-H.; Tokioka, K.; Shimada, I.; Arata, Y. J. Am. Soc. Mass Spectrom. 1996, 7, 707-721.

2718 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

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© 1998 American Chemical Society Published on Web 05/15/1998

Figure 1. Base peak intensity trace of capillary LC/ESI IT-MS of the lysyl-endopeptidase digest of RCM-IgG2b. Peak numbers correspond to those in Table 1. The base peak intensity is 1.4 × 108.

reported that, in the mouse IgG, N-linked oligosaccharides are almost fully fucosylated and lack bisecting GlcNAc.14,15 Kohler et al.16 reported that mouse monoclonal IgG2b contains two forms of the heavy chain that give one major and one minor band with different molecular weights in SDS-PAGE analysis. A similar heterogeneity of the heavy chain has been reported for mouse IgG2b expressed by other cell lines under different culture conditions.17-19 Kohler et al.16 have also revealed that these two types of heavy chain are different in the degree of glycosylation, which can be diagnostic of mouse IgG2b. Kim et al.20 successfully separated mouse monoclonal IgG2b into three phenotypes that are different in the degree of sialylation of the heavy chain. In comparison of the peptide maps of each IgG2b with a different phenotype, they concluded that (1) ∼40% of the heavy chain of the mouse IgG2b antibodies are O-glycosylated at Thr-211AH in the hinge region and (2) three types of tetrasaccharides are predominantly expressed at Thr-221AH, i.e., NeuGc-Gal(NeuGc-)GalNAc-, its isomer, and GalNAc, Gal, NeuGc, and NeuAc. In the present paper, we discuss the result of the use of LC/ ESI IT-MS/MS for the complete peptide and glycopeptide mapping of IgG2b. We have successfully characterized, in addition to the amino acid sequence, the structure of minor O-linked sugar chains at Thr-221AH and N-linked sugar chains at Asn-297H of IgG2b. The strategy we used is as follows. Reduced and carboxymethylated mouse antidansyl monoclonal IgG2b (RCMIgG2b) was digested with lysyl-endopeptidase. Proteolytic peptides were subjected to a capillary HPLC separation and then introduced to an ion trap mass spectrometer. A LC/ESI IT-MS spectrum and a LC/ESI IT-MS/MS spectrum of each peptide was obtained. It was possible to characterize all proteolytic peptides, (14) Mizuochi, T.; Hamako. J.; Titani, K. Arch. Biochem. Biophys. 1987, 257, 387-394. (15) Rothman, R. J.; Warren, L.; Vliegenthart, J. F. G.; Hard, K. J. Biochemistry 1989, 28, 1377-1384. (16) Kohler, G.; Hengartner, H.; Schulman, M. J. Eur. J. Immunol. 1978, 8, 82-88. (17) Demignot, S.; Gaenett, M. C.; Baldwin, R. W. J. Immunol. Methods 1989, 121, 209-217. (18) Fernandez, P.-A.; Ternynck, T.; Avrameas, S. Mol. Immunol. 1989, 26, 539549. (19) Sumii, H.; Tsutsui, K.; Hatsushika, M.; Inoue, H.; Tanabe, G.; Oda, T. Acta Med. Okayama 1989, 43, 135-141. (20) Kim, H.; Yamaguchi, Y.; Masuda, K.; Matsunaga, C.; Yamamoto, K.; Irimura, T.; Takahashi, N.; Kato, K.; Arata, Y. J. Biol. Chem. 1994, 269, 1234512350.

by using the molecular masses of peptides that are generated by proteolysis with specific enzymes and the sequence information obtained by LC/ESI IT-MS/MS. Glycopeptide mapping of IgG2b was carried out for the first time on the basis of the sequencespecific information for N- and O-linked sugar chains obtained by LC/ESI IT-MS/MS. EXPERIMENTAL SECTION Materials. Dithiothreitol, iodoacetic acid, acetonitrile, lysylendopeptidase, and trifluoroacetic acid (TFA) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Staphylococcus aureus V8 protease was purchased from ICN ImmunoBiologicals (Costa Mesa, CA). Preparation and Purification of IgG2b. IgG2b originated from C3H/SW mice (haplotype j) was produced by cultivation of hybridoma cells and purified in the same manner as reported previously.21 Reduction and S-Carboxymethylation. Lyophilized IgG2b (60µg) was dissolved in 60 µL of 0.5 M Tris-HCl buffer (pH 8.1) that contained 6 M guanidine and 2 mM ethylenediaminetetraacetic acid (EDTA). After an addition of 105 µg of dithiothreitol, the mixture was bubbled with nitrogen, heated at 50 °C for 3 h, and allowed to stand overnight at room temperature. To this solution, 500 µg of iodoacetic acid was added; the pH of the solution was kept at pH 8.0-8.5 with 1 N NaOH. After 30 min, the protein was dialyzed against 50 mM ammonium bicarbonate buffer at 4 °C and then lyophilized. Proteolytic Digestion of IgG2b. Lysyl-endopeptidase digestion of reduced and carboxymethylated (RCM) IgG2b (60 µg) was performed under the conditions of a 200:1 weight ratio of substrate to enzyme in 12 µL of 50 mM ammonium bicarbonate buffer (pH 8.4) at 37 °C for 15 h. V8 protease digestion of RCM-IgG (60 µg) was performed under the conditions of a 100:1 molar ratio of substrate to enzyme in 12 µL of 50 mM ammonium bicarbonate buffer (pH 7.9) at 37 °C for 8 h. The hydrolysis was stopped by an addition of 0.1% TFA, and then the solution was lyophilized. Liquid Chromatography/Electrospray Ionization Ion TrapMass Spectrometry. The capillary HPLC system consisted of an ABI 140B dual-syringe solvent delivery system (Applied Biosystems, Inc., Foster City, CA) with a Kontron (Milano, Italy) 433 capillary detector set to detect 210-nm light. The solvents (21) Kato, K.; Matsunaga, C.; Igarashi, T.; Kim, H.; Odaka, A.; Shimada, I.; Arata, Y. Biochemistry 1991, 30, 270-278.

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Table 1. Observed Mass Values in LC/ESI IT-MS of the Lysyl-endopeptidase Digest of RCM-IgG2b with the Attribution of the Mass Numbers to a Previously Reported Sequence13 position peak

no.a

46 5 31 36 44 20 53 16 10 31 34 19 18 15 4 25 49 38 22 23 53 42 51 27 33 4 45 41 48 48 48 9 2 3 32 43 40 50 37 15 15 8 26 24 9 9

H or L L L L L L L L L L L L L L H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

Lb

fromc

toc

obsd massd

expect. ave

∆mf

expect. monoisotopicg

∆mh

1 40 46 51 75 104 108 143 148 150 170 184 200 208 1 4 20 44 52C 65 76 120 148 214 214 228A 229 249 259 259 259 318 321 323 327 339 341 362 393 410 414 417 420 434 439 440

39 45 50 74 103 107 142 147 149 169 183 199 207 214 3 19 43 52B 64 75 119 147 213 228 228 228D 248 258 317 317 317 320 322 326 338 361 361 392 409 413 419 419 433 438 446 446

4919.8 613.4 649.4 2519.0 3222.7 502.3 3730.6 588.3 333.2 2250.6 1535.4 2041.8 832.5 927.4 375.2 1546.4 2808.8 1315.2 1468.6 1282.4 5387.8 2839.4 6839.8 2976.8 1997.8 574.3 2156.6 1116.6 8259.0 8423.5 8586.0 423.3 308.2 474.3 1356.4 2574.6 2333.2 3470.6 1878.6 505.3 778.4 462.5 1651.4 700.4 845.5 717.5

4920.5 613.7 649.9 2519.7 3222.6 502.6 3731.2 588.7 333.4 2250.3 1535.7 2042.1 833.0 928.0 375.4 1546.7 2809.1 1315.5 1468.6 1282.4 5387.1 2839.2 6841.5 2977.2 1998.3 574.6 2156.5 1117.4 8259.0 8422.0 8584.1 423.5 308.6 474.5 1356.6 2575.1 2333.8 3470.8 1879.0 505.7 778.9 462.5 1651.8 700.8 846.0 717.8

-0.7 -0.3 -0.5 -0.7 0.1 -0.3 -0.6 -0.4 -0.2 0.3 -0.3 -0.3 -0.5 -0.6 -0.2 -0.3 -0.3 -0.3 0.0 0.0 0.7 0.2 -1.7 -0.4 -0.5 -0.3 0.1 -0.8 0.0 1.5 1.9 -0.2 -0.4 -0.2 -0.2 -0.5 -0.6 -0.2 -0.4 -0.4 -0.5 0.0 -0.4 -0.4 -0.5 -0.3

4917.4 613.3 649.4 2518.2 3220.5 502.3 3728.8 588.3 333.2 2249.0 1534.7 2040.8 832.5 927.4 375.2 1545.8 2807.2 1314.7 1467.7 1281.7 5383.6 2837.4 6837.3 2975.3 1997.0 574.2 2155.1 1116.6 8253.6 8416.8 8578.9 423.2 308.1 474.3 1355.8 2573.5 2332.4 3468.6 1877.9 505.3 778.4 462.2 1650.8 700.4 845.5 717.4

2.4 0.1 0.0 0.8 2.2 0.0 1.8 0.0 0.0 1.6 0.7 1.0 0.0 0.0 0.0 0.6 1.6 0.5 0.9 0.8 4.2 2.0 2.5 1.5 0.8 0.1 1.5 0.0 5.4 6.7 7.1 0.1 0.1 0.0 0.7 1.1 0.8 2.0 0.7 0.0 0.0 0.3 0.7 0.0 0.0 0.1

attachment

O-linked sugari

N-linked sugar (E)j N-linked sugar (FG) N-linked sugar (H)

a Peak number in Figure 1. b Heavy chain (H) or light chain (L). c The position of the amino acid residue. d Observed mass value in LC/ESI IT-MS calculated by deconvolution. e Expected average mass value. f Mass difference between the observed and expected average mass values. g Expected monoisotopic mass value. h Mass difference between observed and expected monoisotopic mass values. i O-linked glycopeptide. j Nlinked glycopeptide in Figure 7.

consisted of (A) 0.1% aqueous TFA and (B) acetonitrile/water (9: 1) with 0.095% TFA. The elution was 8-min isoclatic with 92% solvent A and then gradient elution of 92% A to 60% A (8-45 min), 60% A to 20% A (45-65 min), 20% A to 10% A (65-80 min), and 10-min isoclatic with 10% A performed for the isolation of peptides on a Vydac C18 capillary column packed by LC Packings (San Francisco, CA). The solvents were mixed and split by an Acurate unit (LC Packings) and the flow rate was reduced to ∼3 µL/min. Electrospray ionization mass spectrometry (ESIMS) was performed on a Finnigan (San Jose, CA) LCQ equipped with a Finnigan electrospray ion source. The electrospray ion source was operated at a potential difference of between 3.0 and 4.5 kV. Nitrogen was used for sheath and auxiliary gas. A 50% methanol 2720 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

solution was used for sheath liquid at a flow rate 3 µL/min. LC/ ESI IT-MS and LC/ESI IT-MS/MS were run in an automated LC/ MS-LC/MS/MS mode that monitored for a signal threshold and performed MS/MS on the base peak when the threshold criterion was exceeded. The ion trap parameters were employed as follows. The trap was run with automatic gain control for all experiments. In this mode, the system automatically selects the trapping parameters to keep the number of ions present in the trap to a constant preset value. The electron multiplier was set to -850 V. The number of “microscans” collected were three and two for full MS and MS/ MS, respectively. In CID mode, the trap was filled for up to 50 ms, depending on the number of ions entering the trap per unit

of time. In this mode, the threshold to trigger ion selection was 5.0 × 104, and the default collision energy was set to 50%. Proteolytic peptides (∼200 pmol) were injected into the capillary HPLC system and then introduced into the mass spectrometer after separation. Peptide Assignment. Molecular weights and partial amino acid sequences of the peptides were attributed to the sequence of RCM-IgG2b using BioWorks software as supplied with the LCQ. RESULTS AND DISCUSSION Amino Acid Sequence of IgG2b. Capillary LC/ESI IT-MS and -MS/MS analyses were applied to the lysyl-endopeptidase digest of RCM-IgG2b to complete the assignment of mass values to the peptides. The analyses were carried out by an automated LC/MS-LC/MS/MS mode that monitored for a signal threshold and performed MS/MS on the base peak when the threshold criterion was exceeded. Figure 1 shows the base peak trace of capillary LC/ESI IT-MS of the lysyl-endopeptidase digest. Protonated molecular masses calculated by the deconvolution of the observed ions in LC/ESI IT mass spectra of the lysyl-endopeptidase digest of RCM-IgG2b are also summarized in Table 1. The amino acid sequence of RCM-IgG2b was totally characterized by the molecular weights of lysyl-endopeptidase digested peptides. Moreover, partial sequences of all the peptides generated by the lysyl-endopeptidase digestion except for the peptide CK (321H322H) and N-glycopeptide (259H-317H) could be successfully confirmed by LC/ESI IT-MS/MS. Mass spectrometric peptide mappings have usually been carried out using only the molecular masses of the peptides and the specificity of the proteases. Ambiguity sometimes remains after these analyses, because it is not possible to assign the molecular mass of a peptide to the amino acid sequence of the original protein when the molecular mass of a peptide can be assigned to more than two positions in the original protein. However, LC/ESI IT-MS/MS can potentially eliminate such ambiguity, because sequence information can also be obtained for a given digest fragment. For example, lysyl-endopeptidase digest can be assigned two peptides whose molecular masses were very close, TDSFSCNVRHEGLKNYYLK (420H-438H) (average molecular mass 2332.6 u) and GLVRAPQVYILPPPAEQLSRK (341H-361H) (average molecular mass 2332.8 u). It was therefore very difficult to distinguish each by using molecular masses of the peptides. It is certainly possible to assign the peptide to the original amino acid sequence of the protein if the peptide is isolated and sequenced by MS/MS or Edman degradation. However, it takes considerable time and a significant quantity of sample to generate enough peptide for analysis after enzymatic cleavage of RCM-IgG2b, since IgG is a very large protein. Development of a new method has been desired for the identification of peptides generated from a large protein. Figure 2 shows a product ion spectrum obtained by LC/ESI IT-MS/MS measurement of peaks 40 in Figure 1. The product ion spectrum in Figure 2 was obtained from the doubly charged ion (m/z 1167.5) of the peptide. The nomenclature used to identify the peptide fragment ions in this paper is based on that by Biemann.22 Singly charged ions of y10 (m/z 1123.4), y11 (m/z 1235.9), y12 (m/z 1349.8), y13 (22) Biemann, K. In Mass Spectrometry; McCloskey, J. A., Ed.; Methods in Enzymology 193; Academic: San Diego, CA, 1990; Chapters 18 and 25, Appendix 5.

Figure 2. Product ion spectrum obtained by LC/ESI IT-MS/MS measurement of peak 40 in Figure 1. The product ion spectrum was obtained from the doubly charged ion (m/z 1167.5) of the lysylendopeptidase-digested peptide GLVRAPQVYILPPPAEQLSRK (341H-361H). The base peak intensity is 1.3 × 107.

Figure 3. Product ion spectrum obtained by LC/ESI IT-MS/MS measurement of peak 6 in Table 2. The product ion spectrum was obtained from the doubly charged ion (m/z 679.1) of V8 proteasedigested peptide ATHKTSTSPIVKS (196L-208L). The base peak intensity is 3.4 × 104. The intensity of the precursor ion (m/z 679.1) was weak, because the peptide was obtained by nonspecific cleavage of V8 protease and the yield of the peptide was not high. The signalto-noise ratio of the product ion spectrum was poor.

(m/z 1512.0), y14 (m/z 1612.9), y16 (m/z 1836.5), and b11 (m/z 1211.6) and the doubly charged ion of c20-2 (m/z 1102.3) clearly fit to the theoretical masses of the product ions calculated from the sequence of GLVRAPQVYILPPPAEQLSRK (341H-361H). Thus it was possible to assign accurately the molecular mass of the peptide to the original protein by LC/ESI IT-MS/MS. March mentioned that the ion trap offers two principal advantages when used in the MS/MS mode.23 First, the ion trap operates in a pulsed mode, in contrast to sector and triple-stage quadrupole instruments, which operate in a continuous mode, so that it can accumulate ions mass selectively over time. In this way, a target ion number can be selected to ensure constant signalto-noise ratio over a wide range of eluent concentrations. Second, (23) March, R. E. J. Mass Spectrom. 1997, 32, 351-369.

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Table 2. Observed Mass Values in LC/ESI IT-MS of the V8 Protease Digest of RCM-IgG2b with the Attribution of the Mass Numbers to a Previously Reported Sequence13 position peak

no.a

32 33 17 23 19 21 5 1 6 11 11 4 8 22 15 28 19 16 7 9 16 14 21 9′ 13 9 18 27 24 2 19 19 19 15 14 6 5 26 24 25 21 8 12

H or L L L L L L L L L L L L H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

Lb

fromc

toc

obsd massd

expect. ave

∆mf

expect. monoisotopicg

∆mh

1 1 106 111 155 155 188 196 196 196 202 209 1 6 7 8 36 43 51 61 74 213 213 217 217 228B 228B 236 273 284 295 295 295 305 319 377 381 390 390 390 400 419 431

79 105 123 143 162 187 195 201 208 214 214 214 6 50 35 17 50 50 61 73 85 228A 228A 228A 228A 235 272 249 283 294 318 318 318 318 333 388 389 418 430 446 418 430 446

9231.4 12092.2 1887.4 3503.2 887.5 3801.4 1068.2 644.2 1357.0 2179.4 1553.4 840.2 746.3 4879.4 2952.9 828.7 1815.0 931.4 1666.0 1381.4 1863.8 2254.2 3232.3 2735.8 1757.0 1227.6 5213.8 1488.8 1334.8 1308.2 4251.4 4412.5 4575.4 1670.0 1791.2 1360.2 1061.2 3402.7 4865.0 6672.2 2285.4 1481.8 1826.0

9231.4 12092.6 1887.1 3503.9 888.0 3802.0 1068.1 644.7 1357.6 2179.4 1553.7 840.9 746.3 4879.4 2954.3 828.9 1815.0 932.1 1665.8 1381.5 1866.2 2255.6 3234.5 2735.8 1757.0 1228.4 5213.0 1488.7 1335.5 1308.4 4251.5 4413.6 4575.8 1670.9 1791.1 1360.4 1061.1 3402.8 4865.4 6672.5 2285.6 1481.6 1826.2

0.0 -0.4 0.3 -0.7 -0.5 -0.6 0.1 -0.5 -0.6 0.0 -0.3 -0.7 -0.5 0.0 -1.4 -0.2 0.0 -0.7 0.2 -0.1 -2.4 -1.4 -2.2 0.0 0.0 -0.8 0.8 0.1 -0.7 -0.2 -0.1 -1.1 -0.4 -0.9 0.1 -0.2 0.1 -0.1 -0.4 -0.3 -0.2 0.2 -0.2

9225.7 12085.0 1886.0 3501.7 887.5 3799.7 1067.4 644.3 1356.7 2178.1 1552.7 840.3 746.4 4876.2 2952.3 828.4 1813.9 931.5 1664.8 1380.7 1865.0 2254.1 3232.4 2734.1 1755.8 1227.5 5209.5 1487.8 1334.7 1307.6 4248.9 4411.0 4573.0 1669.8 1789.9 1359.6 1060.4 3400.6 4862.3 6668.3 2284.1 1480.6 1825.0

5.7 7.2 1.4 1.5 0.0 1.7 0.8 -0.1 0.3 1.3 0.7 -0.1 -0.1 3.2 0.6 0.3 1.1 -0.1 1.2 0.7 -1.2 0.1 -0.1 1.7 1.2 0.1 4.3 1.0 0.1 0.6 2.5 1.5 2.4 0.2 1.3 0.6 0.8 2.1 2.7 3.9 1.3 1.2 1.0

attachment

O-linked sugari O-linked sugar

N-linked sugar (E)j N-linked sugar (FG) N-linked sugar (H)

a Peak number of base peak chromatogram of capillary LC/ESI IT-MS of the V8 protease digest of RCM-IgG2b (The chromatogram is not shown.). b Heavy chain (H) or light chain (L). c The position of the amino acid residue. d Observed mass value in LC/ESI IT-MS calculated by deconvolution. e Expected average mass value. f Mass difference between the observed and expected average mass values. g Expected monoisotopic mass value. h Mass difference between observed and expected monoisotopic mass values. i O-linked glycopeptide. j N-linked glycopeptide in Figure 7.

CID in the ion trap is wrought by several hundred collisions of mass-selected ions with helium buffer gas atoms. Under these conditions, the energy transferred in a single collision is seldom greater than a few vibrational quanta such that the dissociation reaction channels of lowest energy of activation are accessed almost exclusively. This behavior is highly advantageous in analytical chemistry in that total charge is conserved within a single fragment ion species. In addition, high-quality MS/MS spectra can be obtained by ion trap MS even if a constant collision voltage is applied to the doubly and triply charged precursor ions. In the case of triplequadrupole MS, it is necessary to seek optimum MS/MS measurement conditions for each precursor ion with different charge states. Accordingly, ion trap MS shows high capability for MS/MS analysis. 2722 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

One of troublesome problems for the peptide mapping using mass spectrometry is caused by unexpected cleavage by proteases. It has been known that nonspecific cleavage in the tryptic digestion is derived from chymotryptic activity which remained in the non-TPCK-treated enzyme.1,2 In the peptide map of RCMIgG2b prepared by V8 protease digestion, it was not possible to assign a peptide, whose doubly charged molecular ion was observed at m/z 679.1, to any sequence of RCM-IgG2b. Figure 3 shows a product ion spectrum obtained from (M + 2H)2+ of this peptide. Singly charged ions of y5 (m/z 543.0), y6 (m/z 630.3), y7 (m/z 731.2), y8 (m/z 819.0), y9 (m/z 919.3), y10 (m/z 1047.5), b4 (m/z 438.2), b7 (m/z 727.5), b8 (m/z 814.2), b10 (m/z 1024.4), and b11 (m/z 1123.4) correspond to the theoretical masses of the product ions calculated from the sequence of ATHKTSTSPIVKS (196L-208L). This suggested that the C-terminal of this peptide

Table 3. Twelve Types of O-Glycoform Expressed at Thr-221AH of Mouse IgG2b glycopeptidea

glycoform

obsd av mass,f u

rel content,g %

A B Cb Db Eb Fb G H Ic Jc K L M

NeuGc-(NeuGc-)Hex-HexNAc-peptided,e NeuGc-Hex-(NeuGc-)HexNAc-peptided,e NeuGc-Hex-(NeuAc-)HexNAc-peptided,e NeuAc-Hex-(NeuGc-)HexNAc-peptided,e NeuGc-(NeuAc-)Hex-HexNAc-peptided NeuAc-(NeuGc-)Hex-HexNAc-peptided Hex-(NeuGc-)HexNAc-peptided NeuGc-Hex-HexNAc-peptided Hex-(NeuAc-)HexNAc-peptided NeuAc-Hex-HexNAc-peptided Hex-HexNAc-peptided HexNAc-peptided nonglycosylated peptided

1488.8 1488.8 1480.9 1480.9 1480.9 1480.9 1335.6 1335.6 1326.9 1326.9 1181.8 1100.7 999.0

6.8 5.7 0.9 0.7 0.4 0.3 1.8 2.0 0.1 0.1 1.6 0.5 79.2

a Glycopeptides are identified by alphabetical codes corresponding to those of the peaks in the mass chromatogram in Figure 4. b Differenciation between C and D as well as E and F could not be carried out by the product ion mass chromatogram. c Differenciation between I and J could not be carried out. d Amino acid sequence of the peptide is KLEPSGPISTINPCPPCK(214H-228H). e Sugar composition has already been determined; Hex and HexNAc correspond to Gal and GalNAc, respectively.20 f Observed mass is (M + 2H)+ of glycopeptide. g Estimated from the peak area in the mass chromatogram in Figure 4.

Figure 4. Mass chromatograms of 12 types of O-glycoforms at Thr-221AH of mouse IgG2b. O-Glycosylated peptides originated from Lys214H to Lys-228H are observed as doubly charged ions. The sequence of the peptide is KLEPSGPISTINPCPPCK (214H-228H). Monitored mass numbers and estimated sugar chain structures attached to Thr-221AH are listed in Table 3. Alphabetical capital letters in the chromatogram correspond to glycopeptides in Table 3. Insets: (a) Mass chromatograms of m/z 1488.8 and 1335.6, and product ion mass chromatogram of m/z 1254.3. (b) Mass chromatogram of m/z 1480.9 and product ion mass chromatogram of m/z 1335.6, 1326.9, 1254.3, and 1246.5. The manual MS/MS experiment was carried out from m/z 1480.9 as a precursor ion.

was Ser, not Glu or Asp. This would indicate that V8 protease can cleave the Ser residue at the C-terminal. Protonated molecular masses calculated by the deconvolution of the observed ions in LC/ESI IT mass spectra of the V8 protease digest of RCM-IgG2b are also summarized in Table 2. Glyco-Mapping of O-Linked Sugar Chain at Thr-221AH. With a mild trifluoroacetic acid treatment, Kim et al. have shown that the sugar chain of a major glycopeptide is biantennary.20 A sequence of NeuGc-Gal-(NeuGc-)GalNAc- was suggested. In the present study, we were able to shown that 12 types of oligosaccharides exist as O-linked sugar chains. Nine of 12 were newly found in this study. O-Glycosylated peptides originating from Lys-214H to Lys-228H were observed as doubly charged ions. The results by LC/ESI IT/MS/MS analyses are summarized in

Table 3. Figure 4 shows a mass chromatogram of these 12 glycopeptides and the nonglycosylated peptide KLEPSGPISTINPCPPCK (214H-228H). The content of each glycoform was calculated by peak area observed in the mass chromatogram for the first time. A 200-pmol aliquot of the digest was applied to this analysis. This indicates that the doubly charged molecular ion of Hex-(NeuAc-)HexNAc-peptide was observed with only 0.2 pmol of material. Moreover, this represents minor Oglycosylation of mono-, di-, and trisaccharides in IgG2b. The calculated molecular weight of major glycopeptide-containing tetrasaccharides NeuGc-(NeuGc-)Gal-GalNAc- and NeuGc-Gal-(NeuGc-)GalNAc- is 2975.6.20 This shows that peaks A and B in the mass chromatogram of m/z 1488.8 in Figure 4 actually correspond to the doubly charged ions of these glycoAnalytical Chemistry, Vol. 70, No. 13, July 1, 1998

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Figure 5. Product ion spectrum of NeuGc-Gal-(NeuGc-)GalNAc-peptide (214H-228H) generated by lysyl-endopeptidase digestion. The spectrum was obtained by LC/ESI IT-MS/MS measurement from the doubly charged molecular ion (m/z 1489.0) of the Oglycopeptide of peak A of Figure 4. The amino acid sequence of the glycopeptide is KLEPSGPISTINPCPPCK (214H-228H). The base peak intensity is 5.7 × 106.

peptides. Figure 5 shows the product ion spectrum obtained from the doubly charged ion (m/z 1488.8) in peak A in Figure 4. The sugar sequence of NeuGc-Hex-HexNAc was estimated from the mass value of the product ions at m/z 1335.6, 1181.8, 1100.7, and 999.0 in Figure 5. A mass difference of 153.3 between the precursor ion (m/z 1488.8) and m/z 1335.6 corresponds to the half mass value of the NeuGc residue. For the differentiation of these two isomers, it is necessary to determine which sugar residue, Hex or HexNAc, links to NeuGc. Analysis by product ion mass chromatogram was proven very effective for the differentiation of these two isomers; we describe here the technique in detail. If NeuGc is linked to HexNAc, the product ion of m/z 1254.3, which corresponds to the doubly charged ion of a peptide containing NeuGc-HexNAc- derived from the cleavage of the bond between Hex-HexNAc, should be observed in the product ion mass chromatogram. In the product ion mass chromatogram of m/z 1254.3 in inset a of Figure 4, a peak at the same retention time as peak B was clearly observed. Accordingly, it was concluded that peak B corresponded to the glycopeptide containing NeuGc-Hex-(NeuGc-)HexNAc-. Then it was concluded that peak A corresponded to the glycopeptide containing NeuGc-(NeuGc-)Hex-HexNAc-. Taking into account the sugar composition of the glycopeptides,20 it was concluded that peaks A and B corresponded to the glycopeptides containing NeuGc-(NeuGc-)Gal-GalNAc- and NeuGc-Gal-(NeuGc-)GalNAc-, respectively. In the same manner, in the mass chromatogram of m/z 1254.3 in inset a of Figure 4, a small peak at the same retention time as peak G was observed. This result leads to the assignments of peak G to the Hex-(NeuGc-)HexNAc-peptide and, consequently, peak H to the NeuGc-HexHexNAc-peptide. Estimated structures of the glycopeptides containing NeuAc residue are summarized in Table 3. The molecular weight of the glycopeptide composed of GalNAc, Gal, NeuAc, and NeuGc20 is calculated as 2969.6 (average molecular weight). Thus, (M + 2724 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

Figure 6. Product ion spectrum of from the doubly charged ion (m/z 1480.9) of the O-glycopeptide of peak C in Figure 4. The spectrum was obtained by LC/ESI IT-MS/MS measurement. The amino acid sequence of the glycopeptide is KLEPSGPISTINPCPPCK (214H228H). The base peak intensity is 2.4 × 105.

2H)2+ of this glycopeptide must be observed at m/z 1480.9. It was not possible to obtain an LC/MS/MS spectrum of m/z 1480.9 by an automated LC/MS-LC/MS/MS system. Automated MS/ MS analysis was carried out only for the base peak ion obtained in ESIMS, but the doubly charged ion at m/z 1480.9 was not the base beak in LC/MS analysis. A manual MS/MS experiment was carried out from m/z 1480.9. In inset b of Figure 4, four peaks (C-F) were observed in the mass chromatogram of m/z 1480.9. The product ion spectrum of m/z 1480.9 of peak C is shown in Figure 6. Since product ion spectra of m/z 1480.9 of peaks D-F resemble that of peak C, these four glycopeptides seemed to be isomers. The product ion mass chromatogram traced by m/z 1335.3 (NeuGc-Hex-HexNAc-peptide), 1326.9 (Hex-(NeuGc)HexNAc-peptide), 1254.3 (NeuGc-HexNAc-peptide), and 1246.5 (NeuAc-HexNAc-peptide) suggested that the sugar sequences of the glycopeptides in peak C or D are NeuGc-Hex-(NeuAc)GalNAc- or NeuAc-Hex-(NeuGc-)HexNAc- (Figure 4 in inset b). Taking into account the sugar composition of the glycopeptides,20 it was concluded that the sugar sequences of the peptides in peaks C and D were NeuGc-Gal-(NeuAc-)GalNAcand NeuAc-Gal-(NeuGc-)GalNAc-, respectively. In the same manner, we proposed that the sugar sequences of the glycopeptides in peak E or F are NeuGc-(NeuAc-)Hex-HexNAc- or NeuAc-(NeuGc-)Hex-HexNAc-. The relative content of C, as listed in Table 3, was estimated to be 0.9% from the peak area traced by m/z 1480.9 in the mass chromatogram in Figure 4. LC/ESI IT-MS/MS analyses from m/z 1326.9 of peaks I and J gave no product ion; the corresponding total amount of glycopeptides is estimated to be only 0.2 pmol. Accordingly, it was not possible to deduce the structures of peaks I and J. Peaks K, L, and M were easily assigned to Hex-HexNAc-peptide, HexNAc-peptide, and nonglycosylated peptide, KLEPSGPISTINPCPPCK, respectively, by LC/MS/MS spectra. N-Linked Sugar Chain at Asn-297H. The structures of the N-linked oligosaccharides at Asn-297H have been known to be heterogeneous. In the case of IgG2b used in the present study,

Figure 7. Structures of the N-linked oligosaccharides at Asn-297H of IgG2b24 and product ion spectrum from the triply charged ion (m/z 1472.2) of N-glycopeptide DYNSTIRVVSTLPIQHQDWMSGKE (295H-318H). Sugar is linked at Asn-297H. The glycopeptide was obtained by V8 protease digestion of RCM-IgG2b. The base peak intensity is 2.3 × 105.

the structures of the N-linked oligosaccharides were determined previously, as shown in Figure 7.24 Quintuply charged ions of three N-glycopeptides 259H-317H, whose expected average molecular masses listed in Table 1 were 8259.0, 8422.0, and 8584.1, were observed at m/z 1652.6, 1685.5, and 1718.0 in the LC-ESI IT mass spectrum of the lysylendopeptidase digest in peak 48 in Figure 1. It was not possible to obtain MS/MS spectra from these three ions, because the intensities of these ions were too weak to give MS/MS spectra automatically. In addition, the molecular masses of glycopeptides 259H-317H were too large to obtain MS/MS spectra by ion trap mass spectrometry. RCM-IgG2b was digested by V8 protease to obtain N-linked glycopeptides that are smaller than those derived from lysylendoprotease digestion. The V8 protease digest was analyzed by automated LC/MS-LS/MS/MS mode. Figure 7 shows a product ion spectrum from m/z 1471.5 that is (M + 3H)3+ of the glycopeptide DYNSTIRVVSTLPIQHQDWMSGKE (295H-318H). The structure of the glycopeptide is also shown in Figure 7. Two series of residual carbohydrate masses marked with / and // were observed in the spectrum. In the / series, Fuc rather than Gal or GalNAc must be predominantly eliminated. Elimination of Fuc, GlcNAc, Gal, GlcNAC, and Man (m/z 1768.1), Man (m/z 1687.5), Man (m/z 1605.9), GlcNAc (m/z 1504.8), and GlcNAc (m/z 1403.7) was sequentially observed in the spectrum. In the // (24) Takahashi, N.; Ishii, I.; Ishihara, H.; Mori, M.; Tejima, S.; Jefferis, R.; Endo, S.; Arata, Y. Biochemistry 1987, 26, 1137-1144.

series, elimination of GlcNAc, Gal, GlcNAc, and Man (m/z 1841.0), Man (m/z 1759.9), Man (m/z 1678.9), GlcNAc (m/z 1577.2), Fuc (m/z 1504.8), and GlcNAc (m/z 1403.7) was observed. It was not possible to determine the full sequence of the oligosaccharide because the limited mass range of the instrument was m/z1849. CONCLUSIONS Capillary LC/ESI IT-MS/MS has successfully been used for the characterization of a large protein such as IgG (150 ku). It was possible to perform rapid and accurate characterization of the amino acid sequence and the structures of O-linked and N-linked carbohydrates of mouse monoclonal IgG2b. Twelve types of O-glycoforms at Thr221AH in the hinge region in IgG2b have been characterized and complete glycoform mapping was carried out in this study. The results of the present study indicate that not only amino acid sequences but also carbohydrate structures of large glycoproteins, such as IgG, can routinely be characterized using LC/ESI IT-MS/MS. ACKNOWLEDGMENT We thank Prof. L. A. Herzenberg, Stanford University, and Dr. V. T. Oi, Becton Dickinson Immunocytometry Systems, for generously providing us with the switch variant cell lines and sequence data of the VL region of IgG1 used in the present work. Received for review November 4, 1997. Accepted March 25, 1998. AC9712153

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