“One-Pot” Methylation in Glycomics Application: Esterification of Sialic

Anal. Chem. 2007, 79, 3894-3900

“One-Pot” Methylation in Glycomics Application: Esterification of Sialic Acids and Permanent Charge Construction Xin Liu,† Xianyu Li,† Kenneth Chan,† Wei Zou,† Patrick Pribil,‡ Xing-Fang Li,§ Michael B. Sawyer,§ and Jianjun Li*,†

Institute for Biological Sciences, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6, Applied Biosystems/MDS Sciex, Concord, Ontario, Canada, L4K 4V8, and Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada, T6G 2G3

A simple and rapid “one-pot” methylation method to esterify sialic acids and construct a permanent charge was developed for N-linked glycan analysis, which combined complete nonspecific proteolytic digestion and methylation. A mixture of Asn-glycans prepared from Pronase E digestion of the glycoprotein was passed through a cationexchange column to convert carboxylic acids to the Na+ form before being methylated with methyl iodide. Derivatives could be easily purified with a hydrophilic affinity chromatography cartridge. Mass spectrometry analysis was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/TOF. The mass spectrometric data indicated that carboxylic acids were methylated in addition to the formation of a quaternary ammonium in the amino group of asparagine residues. Three model glycoproteins, including ribonuclease B, ovalbumin, and transferrin, were employed to demonstrate the merits of this technique. Results showed that the stabilization of sialic acid was achieved in addition to the formation of a permanent charge. Compared to the analysis of underivatized Nglycans, detection sensitivity improved ∼10-fold. The new technique was further evaluated with glycan profiling of serum transferrin and proved to be a sensitive method for the characterizing protein glycosylation. Posttranslational modifications of proteins play key roles in signal transduction, protein localization, cell-cell interactions, etc. A common and important posttranslational modification is glycosylation, which has been shown to be important in developmental biology, pathogen localization to specific host tissues, cell division, tumor immunology, inflammation, and prion diseases.1 Variation of oligosaccharides conjugated to proteins modulates protein function by altering protein folding, biological lifetime, and recognition of binding partners. Clinical relevance of glycosylation * To whom correspondence should be addressed. Tel: +1-613-990-0558. Fax: 613-952-9092. E-mail: [email protected]. † Institute for Biological Sciences. ‡ Applied Biosystems/MDS Sciex. § University of Alberta. (1) Ohtsubo, K.; Marth, J. D. Cell 2006, 126, 855-867.

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variation has been shown in inherited and nongenetic diseases that are the result of alterations in oligosaccharide structures.2-4 Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become a major technique for carbohydrate analysis.5-9 However, the hydrophilic nature of carbohydrates led to relative low signal intensities in the MALDI spectrum. In addition, the absence of basic sites results in complicated mass spectra due to adduction of alkali metal ions, usually Na+ and K+.10,11 Permanent charge derivatization has been shown to be an effective way to eliminate Na+ and K+ adducts during the ionization/desorption process and improve detection sensitivity.12-14 Shinohara et al.15 reported a series of derivatization reagents that could also suppress ionization of contaminants (e.g., peptides) in addition to improving detection sensitivity. These reagents have a hydrazine functionality to derivatize glycans, together with quaternary ammonium/pyridinium or guanidine functionalities as ionization enhancing groups. Although these methods resulted in significant enhancement in detection sensitivity, they were not applicable to analysis of sialylated oligosaccharides, since sialylated glycans are relatively unstable when analyzed by MALDI-MS. Therefore, numerous attempts have been made to develop derivatization methods for stabilizing sialic acid, including methyl (2) Mechref, Y.; Novotny, M. V. Chem. Rev. 2002, 102, 321-369. (3) Dwek, R. A. Chem. Rev. 1996, 96, 683-720. (4) Durand, G.; Seta, N. Clin. Chem. 2000, 46, 795-805. (5) Nishimura, S.; Niikura, K.; Kurogochi, M.; Matsushita, T.; Fumoto, M.; Hinou, H.; Kamitani, R.; Nakagawa, H.; Deguchi, K.; Miura, N.; Monde, K.; Kondo, H. Angew. Chem., Int. Ed. 2004, 44, 91-96. (6) Liu, X.; McNally, D. J.; Nothaft, H.; Szymanski, C. M.; Brisson, J. R.; Li, J. Anal. Chem. 2006, 78, 6081-6087. (7) Larsen, M. R.; Hojrup, P.; Roepstorff, P. Mol. Cell. Proteomics 2005, 4, 107119. (8) Kurogochi, M.; Matsushita, T.; Nishimura, S. Angew. Chem., Int. Ed. 2004, 43, 4071-4075. (9) An, H. J.; Peavy, T. R.; Hedrick, J. L.; Lebrilla, C. B. Anal. Chem. 2003, 75, 5628-5637. (10) Harvey, D. J. Mass Spectrom. Rev. 2006, 25, 595-662. (11) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227. (12) Kameyama, A.; Kaneda, Y.; Yamanaka, H.; Yoshimine, H.; Narimatsu, H.; Shinohara, Y. Anal. Chem. 2004, 76, 4537-4542. (13) Naven, T. J. P.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1996, 10, 829-834. (14) Okamoto, M.; Takahashi, K.; Doi, T. Rapid Commun. Mass Spectrom. 1995, 9, 641-643. (15) Shinohara, Y.; Furukawa, J.; Niikura, K.; Miura, N.; Nishimura, S. Anal. Chem. 2004, 76, 6989-6997. 10.1021/ac070091j CCC: $37.00

© 2007 American Chemical Society Published on Web 04/06/2007

esterification,16,17 permethylation,2 perbenzolylation,18 and amidation.19 These methods were proposed to derivatize free glycans from glycoproteins released by enzymatic digestion or chemical hydrolysis. Alternatively, structural information of oligosaccharides can be obtained by analyzing glycopeptides.6,7,9 In our previous work, we described a universal glycomics technique that combined complete nonspecific proteolysis digestion and permethylation.6 The higher enzyme-to-protein ratio and longer digestion time produced Asn-linked glycans. Taking advantage of the reactive chemical feature of the amino acid, the glycan can be further derivatized with various functional groups. Introduction of fixed charges in peptides has proved to be useful for increasing detection sensitivity and facilitating interpretation of their mass spectra. A number of reports focused on utilizing labeling reagents containing fixed charge functionalities,20 while only a few papers described direct formation of a quaternary ammonium using methyl iodide.21-24 It was reported that methylation of peptides can be achieved by using methyl iodide in aqueous methanol solution to form a trimethylammonium with a poor yield and many byproducts.21 To overcome these problems, Kaplan’s group developed an in vacuo methylation method that resulted in a quaternary ammonium moiety on the N-terminus of the peptide with an ∼100% yield.22,23 Recent studies indicated that the fixed charge only formed at the N-terminus of peptides without modifying the -amino groups of lysine by controlling the pH of the reaction.24 Although in vacuo methylation has been successfully applied for qualitative and quantitative analysis of proteins and peptides, the complicated sample treatments and long reaction time limit its application. In this paper, we report a novel technique to fix a permanent charge at the amino group and form methyl esters at carboxylic acids. This simple derivatization approach allows us to analyze sialylated glycans without losing terminal sialic acid groups as well as improving analysis sensitivity. MATERIALS AND METHODS Chemicals and Materials. Ribonuclease B (RNase B), ovalbumin (chicken egg white albumin), human transferrin standard, Pronase E, and human serum were purchased from Sigma-Aldrich (St. Louis, MO). Peptide-N-glycosidase F (PNGase F) was obtained from Roche Diagnostics (Indianapolis, IN). Porous graphitic carbon (PGC) cartridges were obtained from Alltech Associates, Inc. (Deerfield, IL). Dimethyl sulfoxide (DMSO), sodium hydroxide, methyl iodide, ethacridine lactate, 2,5-dihy(16) Norgard-Sumnicht, K. E.; Roux, L.; Toomre, D. K.; Manzi, A.; Freeze, H. H.; Varki, A. J. Biol. Chem. 1995, 270, 27634-27645. (17) Powell, A. K.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1996, 10, 10271032. (18) Chen, P.; Werner-Zwanziger, U.; Wiesler, D.; Pagel, M.; Novotny, M. V. Anal. Chem. 1999, 71, 4969-4973. (19) Sekiya, S.; Wada, Y.; Tanaka, K. Anal. Chem. 2005, 77, 4962-4968. (20) Roth, K. D.; Huang, Z. H.; Sadagopan, N.; Watson, J. T. Mass Spectrom. Rev. 1998, 17, 255-274. (21) Kidwell, D. A.; Ross, M. M.; Colton, R. J. J. Am. Chem. Soc. 1984, 106, 2219-2220. (22) Stewart, N. A.; Pham, V. T.; Choma, C. T.; Kaplan, H. Rapid Commun. Mass Spectrom. 2002, 16, 1448-1453. (23) Poon, C.; Kaplan, H.; Mayer, P. M. Eur. J. Mass Spectrom. 2004, 10, 3946. (24) Simons, B. L.; Wang, G.; Shen, R. F.; Knepper, M. A. Rapid Commun. Mass Spectrom. 2006, 20, 2463-2477.

droxybenzoic acid (DHB), 2,4′,6′-trihydroxyacetophenone (THAP), and cellulose microcrystalline (∼50 µm) were obtained from Sigma-Aldrich. All aqueous solutions were prepared using water purified with a Milli-Q purification system (Millipore, Bedford, MA). Acetonitrile (MeCN), butanol, and ethanol were purchased from Burdick & Jackson (Muskegon, MI). Serums of cancer patients with lymphoma were obtained from patients of the Cross Cancer Institute (The study was approved by the Alberta Cancer Board Institutional Review Board.). Extraction of transferrin was based on reported protocols.25,26 Briefly, 50 µL of human serum was treated with ethacridine lactate and followed by two subsequent precipitations with 25% NaCl and saturated ammonium sulfate. The supernatant was dialyzed against 0.1 M Tris-HCl buffer (pH 7.5) before digestion. Digestion with Pronase E. Glycoproteins were dissolved in 0.1 M Tris-HCl buffer (pH 7.5), and Pronase E (enzyme/protein, 2:1-3:1) was added to the solution, which was incubated at 37 °C for 48 h. The reaction mixture was then boiled for 5 min to deactivate the enzyme. The digested glycopeptides were purified using PGC cartridges. Deglycosylation with PNGase F. Glycopeptides digested by trypsin were dried in vacuo and redissolved in 0.1 M sodium phosphate buffer (pH 7.5). The reaction mixture was incubated with PNGase F (10 units) for 24 h at 37 °C. Samples were then boiled for 5 min to stop the reaction, and the released oligosaccharides were purified using PGC cartridges. Purification Using PGC. PGC cartridges were washed with 3.0 mL of 80% (v/v) MeCN containing 0.1% TFA followed by 3.0 mL of water. Glycopeptides obtained by Pronase E digestion or oligosaccharides released by PNGase F were loaded on PGCs and then washed with water (3.0 mL) to remove the buffer and salts. Glycopeptides or oligosaccharides were first eluted with 25% MeCN in 0.1% TFA and then with 50% MeCN in 0.1% TFA. Each fraction was collected and dried for further analysis or processing. Methylation. Typically, a mixture of Asn-glycans prepared from Pronase E digestion of the glycoprotein was passed through an AG50WX8 resin (Na+) column to convert carboxylic acids to the Na+ form. A total of 50 µL of DMSO and 50 µL of methyl iodide were added to lyophilized samples. The mixture was then reacted with methyl iodide with stirring for 5 h at room temperature. After methyl iodide was removed with a stream of nitrogen, residues were purified by hydrophilic affinity chromatography.27 MALDI-TOF and MALDI-TOF/TOF Analysis. Mass spectra were acquired on a Voyager-DE STR mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a pulsed nitrogen laser (337 nm), with a voltage of 20 kV as the accelerating voltage in the positive mode. Tandem mass spectra were obtained using 4800 MALDI-TOF/TOF (Applied Biosystems/MDS Sciex, Concord, Canada). The matrix used was 10 mg/mL DHB in 50% MeCN. Mixtures, 0.1-0.5 µL, of sample and matrix solutions (1:1 by volume) were applied on the MALDI target. (25) Charlwood, J.; Clayton, P.; Keir, G.; Mian, N.; Winchester, B. Glycobiology 1998, 8, 351-357. (26) Butler, M.; Quelhas, D.; Critchley, A. J.; Carchon, H.; Hebestreit, H. F.; Hibbert, R. G.; Vilarinho, L.; Teles, E.; Matthijs, G.; Schollen, E.; Argibay, P.; Harvey, D. J.; Dwek, R. A.; Jaeken, J.; Rudd, P. M. Glycobiology 2003, 13, 601-622. (27) Wada, Y.; Tajiri, M.; Yoshida, S. Anal. Chem. 2004, 76, 6560-6565.

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Figure 1. MALDI-TOF spectra of Asn-glycans from RNase B: complete (a) and incomplete derivatization (b). Sample amounts loaded on target were equivalent to 5 (a) and 10 ng (b) of RNase B. The matrix was 10 mg/mL DHB in 50% MeCN.

RESULTS AND DISCUSSION It has been well documented that carboxylic acid residues of peptides can be methylated in methanolic HCl solution.28 However, this method is not suitable for glycopeptide methylation, since the presence of HCl can result in hydrolysis of the oligosaccharides. Methylation of carbohydrates can be achieved by using methyl iodide, and the reaction greatly depends on the pH. For example, all hydroxyl groups in glycans could be methylated and the amino acid residue would undergo β-elimination at the amino terminal to form a double bond in the Asn residue when a strong base was used in the permethylation procedure.6 Quaternary ammonium is believed to be formed in β-elimination reactions as an intermediate and the influence of the base in determining the amount or mode of elimination.29 On the other hand, methyl esterification of carboxylic acids in oligosaccharides could be achieved with methyl iodide when the samples were converted to their Na+ form.16,17 It is expected that the carboxylic group at glycopeptides may also be methylated using a similar procedure. Therefore, this quaternary ammonium intermediate might be stabilized with a proper medium and provide a new venue for permanent charge derivatization. Analysis of High-Mannose Oligosaccharides. Purified Asnglycans from Pronase E digestion were employed to investigate the direct methylation using conditions similar to that in permethylation procedures without sodium hydroxide. Three model glycoproteins were employed to demonstrate the merits of this technique. RNase B, a glycoprotein with high mannose structure, was first examined, and the obtained mass spectrum is presented in Figure 1a. A series of ions were detected at m/z 1405.6, 1567.6, 1729.5, 1892.5, and 1054.5 as the ester and charge derivatives, (28) Xu, C. F.; Lu, Y.; Ma, J.; Mohammadi, M.; Neubert, T. A. Mol. Cell. Proteomics 2005, 4, 809-818. (29) Coke, J. L.; Cook, M. P., Jr. J. Am. Chem. Soc. 1967, 89, 2779-2780.

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with an adjacent mass of 162 Da. Tandem mass spectrometry experiments clearly indicated that in addition to complete methyl esterification of their carboxylic groups that a permanent charge was constructed in the amino group (data not shown). However, derivatization was incomplete when the Asn-glycans were not fully converted to the Na+ form (Figure 1b). The observed mass spectrum was predominated by two series of ions. The first series consists of ions at m/z 1385.8, 1547.8, 1709.8, and 1871.8, corresponding to Na+ adducts of the methyl ester of carboxylic acid in Asn residues of Asn-GlcNAc2Man5-8. The second series of ions, m/z 1405.8, 1567.6, 1729.5, and 1892.5, corresponded to the desired products. It was also noticed that, without the proper desalting step, the native Asn-glycan may produce multiple alkaline adducts (e.g., Na+, 2Na+) due the existence of carboxylic acid residues (data not shown). These results suggested that the quaternary ammonium could only form when the carboxylic acid was converted to sodium carboxylate. Stability experiments showed that derivatives could be stable for one week at -20 °C, but only for 20 min at room temperature. Although the derivatives are stable enough for MALDI-TOF analysis, samples should be lyophilized and stored at -20 °C before analysis. To investigate its advantage in sensitivity enhancement, quaternary ammonium Asn-glycan derivatives, underivatized Asnglycans, and free glycans were analyzed and obtained MALDITOF mass spectra were presented in Figure 2. The PNGase F-released glycans (Figure 2a) were detected as their sodium adducts (m/z 1257.4, 1419.3, 1581.3, 1743.2, and 1905.2) and potassium adducts of GlcNAc2Man5-9 glycans (m/z 1273.3, 1435.3, 1597.3, 1761.2, and 1921.2). Figure 2b shows the mass spectrum of RNase B obtained with Pronase E digestion. Both Na + and K+ adducts were observed for underivatized Asn-glycans, while only molecular ions were noticed in the spectrum for ester and charge

Figure 2. MALDI-TOF spectra from different sample preparations: (a) glycans released by PNGase F; (b) Asn-glycans digested by Pronase E; (c) derivatized Asn-glycans. The sample loadings were 10 ng equiv to RNase B for (a) and (b), and 1 ng equiv to RNase B for (c). All spectra were acquired with DHB as matrix.

Figure 3. MALDI-TOF spectrum of derivatized Asn-glycans from 50 ng of ovalbumin. 1, Hex3HexNAc2; 2, Hex4HexNAc2; 3, Hex3HexNAc3; 4, Hex5HexNAc2; 5, Hex4HexNAc3; 6, Hex3HexNAc4; 7, Hex6HexNAc2; 8, Hex5HexNAc3; 9, Hex4HexNAc4; 10, Hex3HexNAc5; 11, Hex5HexNAc4; 12, Hex4HexNAc5; 13, Hex3HexNAc6; 14, Hex5HexNAc5; 15, Hex4HexNAc6; 16, Hex3HexNAc7; 17, Hex6HexNAc5; 18, Hex3HexNAc8; 19, Hex4HexNAc8; 20, Hex7HexNAc6.

derivatives (Figure 2c). The signal-to-noise ratios in the spectrum for quaternary ammonium derivatives from 1 ng of RNase B (Figure 2c) were comparable to that obtained for free glycans (Figure 2a) and underivatized Asn-glycans (Figure 2b) from 10 ng of RNase B. The results demonstrated as much as a 10-fold improvement in detection sensitivity. Analysis of High-Mannose and Hybrid Structure Oligosaccharides. The next example included in our study was chicken ovalbumin, which is a well-studied glycoprotein containing high-mannose and hybrid oligosaccharides. As presented in Figure 3, the MALDI mass spectrum of ester and charge derivatives was predominated by the high-mannose type HexnGlcNAc2 (n ) 3-6) and the hybrid structure type HexnHexNAcm (n ) 3-8, m ) 3-6). More than 20 oligosaccharides were identified when 50 ng of ovalbumin was loaded on the target. The mass spectrum also indicated that three high-mannose-type oligosaccharides with

MannGlcNAc2 (n ) 7-9) were not detected as in the previous report.9 However, seven more hybrid structure-type oligosaccharides were detected, including Hex4HexNAc3, Hex4HexNAc4, Hex4HexNAc5, Hex4HexNAc6, Hex6HexNAc5, Hex4HexNAc8, and Hex7HexNAc6. It is worth mentioning that different glycan profiles may be observed for different sources.30 Analysis of Sialylated Glycans. The loss of sialic acid moiety often occurred during the MALDI analysis and led to difficulties in the sialylated oligosaccharides analysis.10 The proposed procedure has proved to be able to methylate the carboxylic group in asparagine residues. It is expected that it can also methylate carboxylic groups in oligosaccharides and stabilize sialic acids for MALDI-TOF analysis. In order to evaluate the usefulness of the new method in analyzing sialylated oligosaccharides, transferrin was employed as a model in this study. This glycoprotein has two potential glycosylation sites and contains major glycoform of two biantennary glycans with a total number of four sialic acid residues.31 It has been reported that the metastability of sialic acid was related to the selection of matrix, detection mode, and laser power.17 Desialylation was also observed in MALDI analysis of Asn-oligosaccharides with DHB as matrix (data not shown). To this end, linear negative mode and “cooler matrix” THAP were employed to bring about the stabilization of sialylated oligosacchardies.32 The Pronase E digest of transferrin was first analyzed in positive ion detection mode (Figure 4a) and negative ion detection mode (Figure 4b), with THAP as a matrix. As expected, the sensitivity achieved in negative mode was higher than that in positive ion detection mode. However, multiple sodium adducts were observed even in negative ion detection mode as m/z 2336.3, 2358.6, and 2381.1, corresponding to [M - H]-, [M + Na - 2H]-, and [M + 2Na - 3H]-, respectively, where M is the molecular mass of Asn-biantennary glycans. Sodium adducts not only resulted in complex mass spectra but also significantly decreased the sensitivity for sialylated oligosaccharide analysis. For comparison, the Asn-sialylated glycans were desialylated by mild acid hydrolysis before analysis. The removal of sialic acids offered higher sensitivity, but resulted in losing structural information (Figure 4c). As described above, several chemical derivatization methods2,16-19 have been employed for the MALDI analysis of sialylated oligosaccharides. Among these methods, methyl esterification is well accepted as the usual protocol for sialic acid stabilization due to its simple and rapid properties. Therefore, sialylated Asn-glycans were treated with methyl iodide after being converted into their sodium salts. As expected, two major ions of m/z 2117.1 and 2422.3, corresponding to monosialylated and disialylated glycans, were observed with a profound increase in peak intensities (Figure 4d). The results demonstrated that the carboxylic acid of sialic acid was methyl esterified; in addition, a fixed charge was formed at the amino group of asparagine. Moreover, the procedure not only improved detection sensitivity but also stabilized the sialic acid residues for MALDI-TOF analysis. Good signal-to-noise ratios can be achieved even when the amount (30) Harvey, D. J.; Wing, D. R.; Ku ¨ster, B.; Wilson, I. B. J. Am. Soc. Mass Spectrom. 2000, 11, 564-571. (31) Bortolotti, F.; De, P. G.; Tagliaro, F. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2006, 841, 96-109. (32) Papac, D. I.; Wong, A.; Jones, A. J. Anal. Chem. 1996, 68, 3215-3223.

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Figure 4. MALDI-TOF spectra of Asn-glycans from transferrin obtained with different matrixes or sample preparations: (a) underivatzied Asn-glycans, linear positive ion mode with THAP as matrix; (b) underivatzied Asn-glycans, linear negative ion mode, with THAP as matrix; (c) desialylated Asn-glycans, reflectron positive ion mode with DHB as matrix; (d-f) derivatized Asn-glycans, reflectron positive ion mode with DHB as matrix. The sample loadings were 100 (a-d), 10 (e), and 1 ng (f) equiv to transferrin.

Figure 5. MALDI-TOF and TOF/TOF analysis of derivatized glycans from transferrin. The selected precursor ions were (b) m/z 2422 and (c) 2440.

of the glycans loaded on target was equivalent to 1 ng of transferrin. Figure 4d-f represent amounts equivalent to 100, 10, and 1 ng of transferrin total protein loaded onto the MALDI target, respectively. To further investigate the fragmentation pattern of ester and charge derivatives, the glycan was methylated using CH3I and 3898 Analytical Chemistry, Vol. 79, No. 10, May 15, 2007

CD3I. The MALDI-TOF/TOF analyses are presented in Figure 5. Figure 5a shows the mass spectrum of a 1:1 mixture of two derivatives, while panels b and c in Figure 5 are the TOF/TOF tandem mass spectra for precursors at m/z 2422.3 and 2440.3, respectively. A mass difference of 18 Da indicated the addition of six methyl groups, i.e., forming a methyl ester with carboxylic

Scheme 1a

a

The fragment ions are assigned according to Domon and Costello nomenclature.34

groups of sialic acids in addition to constructing a permanent charge in the Asn residue. The TOF/TOF data confirmed that four methyl groups were added to the Asn residue, while both sialic acids were esterified (Figure 5b and c). For example, methyl ester forms of sialic acids gave B1 ions at m/z 306.1 for the light isotope or m/z 309.1 for the heavy isotope. Their corresponding B3-type ions are m/z 671.2 and 674.2, respectively. In addition, Z1- and Y1-type ions were detected at m/z 374.1 and 392.1 for the light isotope derivatization and m/z 386.1 and 404.2 for the heavy isotope derivatization. 0,2 X0 fragments for two isotope forms were observed at m/z 272.1 and 284.2, respectively. In addition, series Y-type ions were detected and details are illustrated in Scheme 1.

We also investigated the potential of the proposed method for quantitative analysis. For example, the ion intensity ratio for Asnbiantennary glycan with a combined 1:1 ratio (CD3I derivative/ CH3I derivative) was 1.25, with a relative standard deviation of 8.4% (n ) 5). The results suggested that further improvement for accuracy and reproducibility needs to be explored. Glycan Profiling of Transferrin Serum Samples. Various pathological states result in alterations in the distribution of human serum transferrin glycoforms, which are of great clinic interest.33 (33) del Castillo Busto, M. E.; Montes-Bayon, M.; Blanco-Gonzalez, E.; Meija, J.; Sanz-Medel, A. Anal. Chem. 2005, 77, 5615-5621. (34) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

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standard (Figure 4d). The unknown peak at m/z 2479.7 might be an impurity introduced from the transferrin extraction procedure. While three cancer patient samples had the same profile as that of the pooled sample, two additional glycoforms were detected in one cancer patient (Figure 6b). The structures were derived as triantennary (m/z 3092.4) and fucosylated triantennary (m/z 3238.5) glycans. Although a detailed understanding of the difference awaits further study, a plausible potential for glycomics is demonstrated.

Figure 6. MALDI-TOF spectrum of derivatized Asn-glycans from transferrin from (a) normal human serum and (b) cancer patient serum.

For example, transferrin has become the most sensitive and specific marker for congenital disorders of glycosylation and chronic alcohol abuse. Therefore, human serum samples were used for further evaluation. In this application, transferrin was isolated from human serum samples using the previously reported approach.25,26 The analysis results for a pooled human serum sample (purchased from Sigma) and from a cancer patient are presented in Figure 6a and b, respectively. A major ion of m/z 2422.7, corresponding to disialylated glycans, was observed together with a weak peak at m/z 2117.5. The mass spectrum of serum transferrin is similar to that obtained from the transferrin

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CONCLUSIONS The data in this study show that carboxylic acids were methylated in addition to the formation of quaternary ammonium in the amino group of asparagine residues, when Asn-glycans were converted to the Na+ form. Merits of this technique are stabilization of sialic acids and enhanced sensitivity as a result of introducing of a permanent charge. It is also expected that the proposed procedure may extend its application to proteomics analysis, since the in vacuo methylation method needs complicated sample treatment and long reaction times.22,23 ACKNOWLEDGMENT The authors thank Dr. Eleonora Altman and Mr. Jacek Stupak for assistance and helpful discussions. X.L. is supported by the NRC Genomics and Health Initiative. Received for review March 9, 2007. AC070091J

January

15,

2007.

Accepted