Chemical Release of O-Glycans Allowing MS Analysis at

Oct 29, 2009 - (17-21) High sensitivity is particularly needed in a search for glycan disease biomarkers, when ..... The MALDI mass spectrum of O-glyc...
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Anal. Chem. 2009, 81, 9546–9552

Articles Enzymatic/Chemical Release of O-Glycans Allowing MS Analysis at High Sensitivity John A. Goetz,†,‡ Milos V. Novotny,*,†,‡,§ and Yehia Mechref*,†,‡,§ Department of Chemistry, National Center for Glycomics and Glycoproteomics, and METACyt Biochemical Analysis Center, Indiana University, Bloomington, Indiana 47405 As the role of O-linked oligosaccharides have been demonstrated to be increasingly important in numerous medical conditions, it is imperative to develop new techniques allowing their analysis at high sensitivity. While mass spectrometry (MS) provides adequate measurements of important O-linked oligosaccharides glycans and their profiles, the release from glycoproteins has not been sufficiently addressed for the needs of biomedical applications. This work describes a new strategy, involving the combination of a complete enzymatic degradation with a chemical release during the solid-phase permethylation of O-linked oligosaccharides. The analytical data implicate highly effective cleavage from the serine and threonine (but not arginine) residues, during permethylation. Tandem MS analyses confirmed these observations for model glycoproteins. Comparative measurements through isotopic labeling MS show this approach to be vastly superior over the previously used cleavage procedures. The ubiquity and biological importance of glycosylated proteins in nature have recently made the glycoprotein structural analysis a desirable focus of numerous studies. From microbes to the highly organized mammalian systems, protein glycosylation is being utilized for the distinct roles in host-pathogen interactions, cellular communication, immunosurveillance, and receptor facili* Corresponding authors. E-mail: [email protected] (Y.M.); novotny@ indiana.edu (M.V.N.). Address: Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN 47405 (Y.M and M.V.N.). † Department of Chemistry. ‡ National Center for Glycomics and Glycoproteomics. § METACyt Biochemical Analysis Center. (1) Calarese, D. A. Science 2003, 300, 2065–2071. (2) Clark, G. F.; Dell, A.; Morris, H. R.; Patankar, M.; Oehninger, S.; Seppala, M. Mol. Hum. Reprod. 1997, 3, 5–13. (3) Pochec, E.; Litynska, A.; Amoresano, A.; Casbarra, A. Biochim. Biophys. Acta 2003, 1643, 113–123.

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tated functions at the membrane level, among other processes.1-5 Many human pathological conditions, including hereditary disorders,6,7 muscular dystrophies,8 neurological diseases,9 cardiovascular and liver disorders,10,11 and different forms of cancer,12,13 have been associated with unusual forms of glycosylation. These associations may occur through the existence of structurally uncommon glycan (oligosaccharide) entities or quantitative alterations in a highly complex repertoire of the more conventional N-linked and/or O-linked glycoconjugate structures. The complex relationships of glycan structure and function necessitate the use of highly sensitive, quantitatively accurate, and informative analytical methods and instrumentation. This is recentlyreflectedinthedevelopmentsinmicroarraytechnologies14-16 and mass spectrometry (MS)-based measurements.17-21 High sensitivity is particularly needed in a search for glycan disease (4) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370–2376. (5) Zachara, N. E.; Hart, G. W. Chem. Rev. 2002, 102, 431–438. (6) Freeze, H. H.; Aebi, M. Curr. Opin. Struct. Biol. 2005, 15, 490-498. (7) Marquardt, T.; Denecke, J. Eur. J. Biochem. 2003, 162, 359–379. (8) Muntoni, F.; Torlli, S.; Brockington, M. Neurotherapeutics 2008, 5, 627– 632. (9) Lefebvre, T.; Guinez, C.; Dehennaut, V.; Beseme-Dekeyser, O.; Morelle, W.; Michalski, J. C. Expert Rev. Proteomics 2005, 2, 265–275. (10) Fu ¨ lo ¨p, N.; Mason, M. M.; Dutta, K.; Wang, P.; Davidoff, A. J.; Marchase, R. B.; Chatham, J. C. Am. J. Physiol. Cell. Physiol. 2007, 292, C1370–1378. (11) Callewaert, N.; Van Vlierberghe, H.; Van Hecke, A.; Laroy, W.; Delanghe, J. Nature Med. 2004, 10, 429–434. (12) Dennis, J. W.; Granovsky, M.; Warren, C. E. Bioassays 1999, 21, 412–421. (13) Lowe, J. B.; Marth, J. D. Annu. Rev. Biochem. 2003, 72, 643–691. (14) Hirabayashi, J. J. Biochem. 2008, 144, 139–147. (15) Liang, P. H.; Wu, C. Y.; Greenberg, W. A.; Wong, C. H. Curr. Opin. Chem. Biol. 2008, 12, 86–92. (16) Paulson, J. C.; Blixt, O.; Collins, B. E. Nat. Chem. Biol. 2006, 2, 238–248. (17) Dell, A.; Morris, H. R. Science 2001, 291, 2351–2356. (18) Geiser, H.; Silvescu, C.; Reinhold, V. Sep. Methods Proteomics 2006, 321– 343. (19) Harvey, D. J. Int. J. Mass Spectrom. 2003, 226, 1–35. (20) Mechref, Y.; Novotny, M. Chem. Rev. 2002, 102, 321–369. (21) Novotny, M.; Mechref, Y. J. Sep. Sci. 2005, 28, 1956–1968. 10.1021/ac901363h CCC: $40.75  2009 American Chemical Society Published on Web 10/29/2009

biomarkers, when only small volumes of physiological fluids are available22-27 from multipurpose clinical studies. During the recent past, methodological difficulties were the primary limitations for developing the field of analytical glycobiology. Among the key limitations has been to achieve the release of oligosaccharides from glycoprotiens, at microscale, with an analytically acceptable reproducibility and without formation of procedural artifacts. With the asparagine-linked (N-linked) glycans, the release of oligosaccharide chains from the polypeptide backbone is now quite easily accomplished with the widely used N-glycanase enzymes, of which the peptide-N-glycosidase F (PNGase F) is the most frequently applied due to its broad28,29 specificity. Alternatively, or additionally, other endoglycosidase enzymes can be employed with some “more difficult” glycans, primarily those derived from plant or insect origin.28 In contrast, a release of O-glycans (serine- or threonine-linked oligosaccharides) from glycoproteins is basically hindered by the unavailability of a broad-specificity enzyme of the likes of N-glycanase hydrolases. The alternative chemical cleavage methods for removal of O-glycans from glycoproteins have been sought in the field of carbohydrate chemistry for many years. Starting with the so-called β-elimination (also known as Carlson’s procedure)30 in a highly alkaline medium, several modifications of this approach have been added more recently.31,32 The major drawback of the original cleavage procedure is that it requires an excessive cleaning step to remove the high salt content which, unfortunately, results in significant sample losses. It is, thus, unsuitable for use at microscale and the following high-sensitivity determinations. Nand O-linked glycans can also be chemically released from glycoproteins with hydrazine.33,34 It has been claimed that Oglycans can be released at 60 °C, while N-glycans necessitate 95 °C for a differential analysis.33 This simple approach has not been generally corroborated by many other laboratories. Additionally, hydrazinolysis presents other difficulties, such as the need for a deacetylation step, which eliminates the information pertaining to the acylation or glycolylation of the sialic acid residues or other undesirable structural alterations of the released oligosaccharides. More recent approaches to β-elimination were introduced by our laboratory,31,32 offering milder reaction conditions and more (22) An, H. J.; Dodds, E. D.; Lebrilla, C. B. Am. Biotechnol. Lab. 2007, 25, 28–29. (23) An, H. J.; Peavy, T. R.; Hedrick, J. L.; Lebrilla, C. B. Anal. Chem. 2003, 75, 5628–5637. (24) Goldman, R.; Ressom, H. W.; Varghese, R. S.; Goldman, L.; Bascug, G.; Loffredo, C. A.; Abdel-Hamid, M.; Gouda, I.; Ezzat, S.; Kyselova, Z.; Mechref, Y.; Novotny, M. V. Clin. Cancer. Res. 2009, 15, 1808–1813. (25) Kyselova, Z.; Mechref, Y.; Al Bataineh, M. M.; Dobrolecki, L. E.; Hickey, R. J.; Vinson, J.; Sweeney, C. J.; Novotny, M. V. J. Proteome Res. 2007, 6, 1822–1832. (26) Kyselova, Z.; Mechref, Y.; Kang, P.; Goetz, J. A.; Dobrolecki, L. E.; Sledge, G. W.; Schnaper, L.; Hickey, R. J.; Malkas, L. H.; Novotny, M. V. Clin. Chem. 2008, 54, 1166–1175. (27) Mechref, Y.; Hussein, A.; Bekesova, S.; Pungpapong, V.; Zhang, M.; Dobrolecki, L. E.; Hickey, R. J.; Hammoud, Z. T.; Novotny, M. V. J. Proteome Res. 2009, 8, 2656–2666. (28) O’Neill, R. A. J. Chromatogr., A 1996, 720, 201–215. (29) Tarentino, A. L.; Plummer, T. H. Method Enzymol. 1994, 230, 44–57. (30) Carlson, D. M. J. Biol. Chem. 1968, 243, 616–626. (31) Huang, Y.; Konse, T.; Mechref, Y.; Novotny, M. V. Rapid Commun. Mass Spectrom. 2002, 16, 1199–1204. (32) Huang, Y.; Mechref, Y.; Novotny, M. V. Anal. Chem. 2001, 73, 6063–6069. (33) Patel, T.; Bruce, J.; Merry, A.; Bigge, C.; Wormald, M.; Jaques, A.; Parekh, R. Biochemistry 1993, 32, 679–693. (34) Takasa, S.; Misuochi, T.; Kobata, A. Methods Enzymol. 1982, 83, 263–268.

convenient ways to remove the residual reagents form small sample amounts. The ammonia-based elimination procedures, leading to either alditols31 or oligosaccharides with the intact reducing end,32 were found satisfactory as a prelude to MS measurements and further tagging chemistries. Under these milder reaction conditions, the so-called “peeling reactions” were significantly reduced. The procedural simplicity of this approach and its effectiveness at microscale levels may substantially aid the efforts in glycoconjugate research. While the newer O-glycan release methodologies31,32 represent a considerable improvement from the past practice, even greater procedural sensitivity is needed in today’s tasks of analytical glycobiology such as the recent efforts to identify glycan biomarkers of human diseases. Specifically, extending any cleavage methodology in the area to submicrogram quantities of isolated glycoproteins or their mixtures should represent an important advance. Based on our brief observations of the different cleavage rates for asparagine-linked and serine/threonine-linked structures on the surface of sodium hydroxide during the permethylation of glycoconjugate samples,35-37 we have developed a new enzymatic/ chemical cleavage scheme for O-linked glycans. This approach involves first the nonspecific proteolysis using the enzyme pronase, followed by our previously described solid-phase permethylation,35-37 in which the free forms of nonreduced oligosaccharides are being formed and immediately converted to the O-linked permethylated derivatives. This combination of the enzymatic and chemical procedures results in a substantial improvement in sensitivity and analytical reproducibility through minimizing sample losses during the treatment. Moreover, this approach extends the cleavage protocols to large glycoproteins where small oligosaccharides could be not accessible during a conventional chemical treatment. MATERIALS AND METHODS Chemicals and Materials. Sodium hydroxide, 20-40 mesh beads, iodomethane-d1, iodomethane-d2, iodomethane-d3, 2,5dihydroxybenzoic acid (DHB), and acetonitrile were acquired from Aldrich (Milwaukee, WI). Chloroform and dimethylsulfoxide (DMSO) were obtained from EM Science (Gibbstown, NJ). Borane-ammonia complex, proteomics-grade trypsin, bovine serum fetuin, human IgA, and 28% aqueous ammonium hydroxide were acquired from Sigma Co. (St. Louis, MO). Pronase was obtained from Roche Applied Science (Mannheim, Germany). The isolated bile salt-stimulated lipase (BSSL) from human milk was kindly provided by Dr. Peter Pahlsson, Department of Clinical Chemistry, University Hospital, Linkoping, Sweden. Digestion with Pronase. The analyzed glycoproteins were dissolved in water to a final concentration of 2 µg/µL, while Pronase was added to a final concentration of 0.2 µg/µL. The reaction mixture was then incubated at 55 °C for 48 h, unless otherwise specified. (35) Kang, P.; Mechref, Y.; Klouckova, I.; Novotny, M. V. Rapid Commun. Mass Spectrom. 2005, 19, 3421–3428. (36) Kang, P.; Mechref, Y.; Kyselova, Z.; Goetz, J. A.; Novotny, M. V. Anal. Chem. 2007, 79, 6064–6073. (37) Kang, P.; Mechref, Y.; Novotny, M. V. Rapid Commun. Mass Spectrom. 2008, 22, 721–734.

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Digestion with Trypsin. The glycoproteins were dissolved in water to a final concentration of 2 µg/µL and trypsin was added to a final ratio of 20:1 (protein/enzyme). The reaction mixture was then incubated at 37 °C overnight. Modified and Conventional β-Elimination of O-linked Oligosaccharides. Glycoproteins tryptically digested, as described above, were subjected to our modified β-elimination protocol.31 The proteolytic peptides were first dried using a vacuum CentriVap Concentrator (Labconco Corporation, Kansas City, MO) prior to the addition of a small volume of 5 µg/µL ammonia-borane complex, prepared in 28% ammonium hydroxide. A 1 µL aliquot of the ammonia-borane complex solution was added to proteolytic peptides originating from the equivalent of 1 µg of glycoprotein. Samples were then incubated for 24 h at 60 °C and subsequently allowed to cool to room temperature prior to the addition of sufficient 1 M HCl to neutralize any residual ammonia-borane complex and ammonium hydroxide. This step was conducted in an ice bath. The samples were dried, methanolwashed, and then dried three times to remove residual borate. The resulting reduced glycans were subsequently subjected to our solid-phase permethylation.35-37 For comparison, the same amount of glycoprotein was also subjected to a conventional β-elimination protocol.30 Briefly, a 5 µg aliquot of glycoprotein was suspended in a 10 µL aliquot of 1.0 M sodium borohydride prepared in 0.1 M aqueous sodium hydroxide. The reaction mixture was then incubated at 42 °C for 18 h. Next, the reaction mixture was placed in an ice bath, while neutralization of the excess sodium borohydride was achieved by adding 1.0 M cold aqueous hydrochloric acid solution until the hydrogen gas evolution ceased (pH ∼ 3-5). Finally, the sample was purified using activated graphite cartridges (Harvard Apparatus, Holliston, MA) as previously described.26,36 Briefly, samples were passed over activated charcoal microcolumns which were preconditioned with 1 mL of acetonitrile (ACN) and 1 mL of 0.1% trifluoroacetic acid (TFA) aqueous solution, as recommended by the manufacturer. After applying the sample, the microcolumn was washed with 1 mL of 0.1% TFA aqueous solution. The samples were then eluted with a 1 mL aliquot of 50% ACN aqueous solution containing 0.1% TFA. Finally, the purified and reduced glycans were evaporated to dryness using a vacuum CentriVap Concentrator prior to solid-phase permethylation. Permethylation. Solid-phase permethylation was performed using our spin-column method.35,37 Briefly, samples were first dissolved in a 90 µL aliquot of DMSO, a 2.7 µL aliquot of water, and a 35 µL aliquot of iodomethane or one of its deuteriated forms. Samples were then passed eight times over a spin-column packed with sodium hydroxide mesh beads. The columns were then washed once with ACN prior to the addition of 400 µL of chloroform. A 1 ml aliquot of 500 mM NaCl aqueous solution was then added prior to liquid-liquid extraction. The addition of sodium chloride solution was repeated twice. The chloroform layer containing permethylated glycans was then dried using a CentriVap Concentrator (Labconco Corporation, Kansas City, Missouri), while the extracted material was resuspended in 4 µL of a 50:50 water/methanol solution prior to MS analysis. MALDI-TOF Analyses. The permethylated samples were spotted and mixed with equal volumes of the sample and a DHB matrix on a MALDI plate. DHB matrix was prepared at a 10 mg/ 9548

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mL concentration in 1 mM sodium actetate aqueous solution. The inclusion of sodium acetate is to promote a nearly complete sodium adduct formation in MALDI-MS. The MALDI plate was then dried under vacuum to ensure uniform crystallization. Mass spectra were acquired using the Applied Biosystems 4800 MALDI TOF/TOF Analyzer (Applied Biosystems Inc., Framingham, MA). This instrument is equipped with Nd:YAG laser operating at 355 nm wavelength. MALDI-spectra were recorded solely in the positive-ion mode, since permethylation eliminates the negative charge normally associated with sialylated glycans. RESULTS AND DISCUSSION We pursued here a new approach to the release and analysis of O-linked glycans, aiming at a substantial improvement over the existing methodologies. High molecular weight proteins have the ability to mask their small O-linked oligosaccharides making the strictly chemical cleavage procedures somewhat inefficient. In the current absence of enzymes cleaving nonspecifically O-linked glycans, it is difficult to ensure high-quality data from chemical procedure alone. Moreover, all existing chemical cleavage procedures are inherently flawed with some undesirable side products and reactions. The approach described here is quite different, capitalizing on the advantages of solid-phase permethylation in conjunction with a complete enzymatic disintegration of the glycoproteins to effectively release and simultaneously permethylate O-glycans. Permethylation is now routinely utilized by many laboratories to simultaneously stabilize the glycans containing labile monosaccharides, such as sialic acids and to enhance sensitivity of MS measurements. The approach reported by our laboratory35-37 employs permethylation directly on the sodium hydroxide beads. The inherently high alkalinity of this derivatization protocol is capable of initiating β-elimination chemistry, thus releasing glycans. In order to evaluate the ability of permethylation conditions to preferentially release O-linked oligosaccharides, we initially subjected intact proteins to the permethylation protocol. The MALDI mass spectrum of permethylated O-glycans derived from intact fetuin glycoprotein is shown in Figure 1a, illustrating a cleavage of protein-bound glycans. Two of the observed ions matched the known fetuin O-glycans (m/z 879.3 and 1240.5) which both possessed free reducing end. The composition of those ions were confirmed through both collision-induced dissociation (CID) and postsource decay (PSD) tandem MS (data not shown). However, the other O-glycan, known to be commonly associated with fetuin (m/z 1689.9), was detected at very low intensity. Although numerous N-glycan structures are also commonly associated with fetuin, we were unable to detect any of them in the permethylated intact fetuin, as shown in Figure 1b. Accordingly, permethylation of intact glycoprotein induces only the release of O-glycans. The release of O-glycans with free-reducing-end from the serine or threonine residues is believed to take place according to the reaction mechanism illustrated in Figure 2. The basic conditions of the permethylation reaction initiate β-elimination of the hydrogen on the R-carbon of either serine or threonine. This reaction then results in creating a double bond between the R-carbon and β-carbon of serine or threonine. This rearrangement further results in the bond cleavage between the β-carbon and the oxygen on the reducing end of the O-linked oligosaccharide,

Figure 1. MALDI-TOF mass spectra of O-glycans released from 5 µg of intact bovine fetuin (a). Intact fetuin glycoprotein was subjected to permethylation and the released O-glycans were purified by liquid-liquid extraction. The high mass range (b) was examined to determine whether N-glycans were released during the procedure; none were detected.

Figure 2. Reaction scheme for the base hydrolysis of O-glycans from a single amino acid.

thus completing the overall β-elimination process. This cleavage creates an alkoxide conjugate base in the presence of high levels of sodium hydroxide. This alkoxide conjugate base undergoes further reaction in the presence of methyl iodide, thus resulting in the permethylation of the free reducing end of O-glycans. Proteolytic Digestion of Glycoproteins. Although permethylation of the intact glycoprotein appeared to result in some release

of O-linked oligosaccharides, the efficiency of this release appears quite low, as suggested by both a weak signal and the inability to detect the third O-glycan structure associated with fetuin which is known to exist at a trace level. We believe this inefficient release of O-glycans is mainly attributed to the three-dimensional structure of the glycoprotein in which some O-glycosylation sites are masked inside. Therefore, they are not directly exposed to the Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

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Table 1. Repeatability of the Enzymatic/Chemical Release of O-Glycans (N ) 6) mass 879.4 1240.6 1689.8 518.4 879.4 967.6 1328.8 1689.8

Figure 3. MALDI-TOF spectra of permethylated O-glycans derived from (a) 0.5 µg of fetuin and (b) 2.5 µg of IgA. Samples were first digested with Pronase for 48 h and then subjected to spin-column permethylation. Marked peaks are impurities originating from sample preparation.

sodium hydroxide beads. This problem could be overcome by first disintegrating the three-dimensional structure of glycoproteins through the use of proteolytic enzymes, such as pronase. This enzyme was previously used in the characterization of N-glycans derived from either standard proteins or bacteria.22,23 For our digestion, we utilized pronase/protein ratios in the range of 1:1 to 1:10, as described in the previously published methods.22,23 The whole pronase reaction mixture was then subjected to a solidphase permethylation after drying. As shown in Figure 3a, the combination of proteolytic digestion and permethylation resulted in a successful release and permethylation of O-glycans derived from a 500 ng aliquot of calf serum fetuin. The spectrum depicted in Figure 3a resulted from the MS analysis of only 10% of the sample (O-glycans derived from ca. 50 ng fetuin). The ions observed in the spectrum (Figure 3a) corresponded to all known O-glycans associated with calf serum fetuin, including the least abundant structure which was only observed without proteolytic digestion at very low intensity (m/z 879.6, 1240.8, and 1689.9). The intensities of the fetuin O-glycans observed in Figure 3a are in agreement with the previously published results.31,32 This result demonstrates that the digestion amply facilitates a full cleavage of O-glycans from the amino acid via the above-mentioned reaction mechanism. The data also support the notion that the three-dimensional structure of this glycoprotein masks some O-glycosylation sites when a chemical release is used alone. Human IgA was also utilized as a model glycoprotein of a substantially larger molecular size to evaluate the effectiveness of the described enzymatic/chemical release method. We have performed the same analysis using an equal molar amount of IgA as used previously for calf serum fetuin. The MALDI mass spectrum of O-glycans derived from IgA using the above-described method is shown in Figure 3b. In this figure, ions with m/z values of 518.3, 879.5, 967.6, 1328.8, and 1689.7 were observed, which also correspond to the known human IgA O-glycans. This new 9550

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average

standard deviation

RSD [%]

Permethylated O-Glycans Derived from Fetuin 77.28 1.43 1.85 20.83 1.37 6.59 1.89 0.30 15.67 Permethylated O-Glycans Derived from IgA 51.38 2.85 44.34 2.75 2.26 0.27 1.06 0.09 0.95 0.25

5.56 6.19 11.72 20.62 26.36

technique, thus, allowed the cleavage and detection of more O-glycans than what has been previously reported.38,39 Repeatability of the described enzymatic/chemical method was demonstrated by comparing the results of six identical analyses. The comparison was based on evaluating the relative signal intensities of six side-by-side analyses. The average value of the relative intensities, their standard deviations, and the relative standard deviations for the generated data are summarized in Table 1. The data demonstrate acceptable standard deviations, especially for the highly abundant O-glycans, suggesting an analytically acceptable repeatability of the described approach. Thus far, we have demonstrated that this technique is both robust and reliable. The above-described results were based on 48 h proteolytic digestion which was determined to be necessary to attain efficient release (data not shown). Comparing the Enzymatic/Chemical Method to Other O-Glycan Cleavage Methods. Our MC-GlycoMAP36 approach allows a direct comparison of the enzymatic/chemical method producing reducing O-glycans, the β-elimination methods (conventional and modified) producing reduced O-glycans, and a modified β-elimination involving trypsin also producing reduced O-glycans. This comparison was based on utilizing equal aliquots (5 µg) of calf serum fetuin (triplicates) subjected to the four different protocols and isotopically permethylated. This amount is the minimum amount needed for the chemical methods. However, the new method described here is capable of quantitive and efficient release of O-glycan from 10-fold lower amounts as shown in Figure 3. The first set of samples was subjected to the enzymatic/chemical protocol, described above, and permethylated using methyl iodide-d0 (CH3I). The second set of samples was subjected to β-elimination using sodium borohydride while being permethylated with methyl iodide-d1 (CH2DI). The third set of samples was subjected to a modified β-elimination using an ammonia-borane complex, while the resulting O-glycans were permethylated using methyl iodide-d2 (CHD2I). The last set of samples was subjected to a modified method of the β-elimination procedure using an ammonia-borane complex, in which the samples were first digested using trypsin and then resuspended in a solution of ammonia-borane complex. This final set of O-linked oligosaccharides was permethylated using methyl iodide-d3 (CD3I). The new approach described here is (38) Renfrow, M. B.; Cooper, H. J.; Tomana, M.; Kulhavy, R.; Hiki, Y.; Toma, K.; Emmett, M. R.; Mestecky, J.; Marshall, A. G.; Novak, J. J. Biol. Chem. 2005, 280, 19136–19145. (39) Tarelli, E.; Smith, A. C.; Hendry, B. M.; Challacombe, S. J.; Pouria, S. Carbohydr. Res. 2004, 339, 2329–2335.

the only approach producing reducing O-glycans, while all the other approaches are producing reduced O-glycans. All sets were then pooled and subjected to MALDI-MS analyses. As depicted in Figure 4, the resulting O-glycans from calf serum fetuin indicate that the enzymatic/chemical methods yield significantly higher signals, which are still consistent with the known levels of these O-glycans, when compared to either the conventional β-elimination or both forms of the modified β-elimination method.30-32 The enzymatic/chemical method utilizing pronase and permethylation of the pronase digest without any purification step demonstrated substantially higher yields (80-95% higher) than any of the other approaches. The efficiency of the release of O-glycans employing the enzymatic/chemical approach was 85% higher than the other approaches in the case of the most abundant

Figure 4. MALDI-TOF mass spectra for C-GlycoMAP analysis of O-glycans, comparing the different chemical release approaches. O-Glycans were cleaved from 5 µg of fetuin through 48 h of Pronase E digestion followed by spin-column permethylation with CH3I; conventional β-elimination followed by spin-column permethylation with CDH2I; β-elimination using ammonia-borane complex followed by spin-column permethylation with CD2HI; β-elimination using ammonia-borane complex after tryptic digestion of the intact fetuin, followed by spin-column permethylation with CD3I.

Figure 5. MALDI-TOF mass spectra of permethylated O-glycans released from a 10 µg aliquot of BSSL, prepared under the optimized conditions described in this work.

Table 2. Composition of BSSL O-Glycan Structures Shown in Figure 5 and Identified in Our Previously Published Work44 obs. mass calc. mass ∆mass 967.487 1141.555 1315.65 1328.654 1345.648 1416.708 1489.706 1502.740 1590.788 1764.873 1777.852 1794.897 1865.922 1938.968 1951.951 2039.989 2126.046 2214.094 2300.117 2388.139 2401.167 2489.180 2562.169 2575.224 2588.056 2663.267 2749.292 2837.277 2850.324 2938.385 3011.371 3112.365 3125.429 3198.425 3211.492 3286.418 3372.505

967.484 1141.573 1315.663 1328.658 1345.673 1416.710 1489.752 1502.747 1590.800 1764.889 1777.884 1794.899 1865.936 1938.978 1951.973 2040.026 2126.062 2214.115 2300.152 2388.204 2401.199 2489.252 2562.047 2575.289 2588.284 2663.341 2749.378 2837.430 2850.425 2938.478 3011.519 3112.567 3125.562 3198.604 3211.653 3286.655 3372.691

0.003 0.018 0.013 0.004 0.025 0.002 0.046 0.008 0.012 0.016 0.032 0.002 0.014 0.01 0.022 0.037 0.016 0.021 0.035 0.065 0.032 0.072 0.122 0.065 0.228 0.074 0.086 0.153 0.101 0.093 0.148 0.201 0.133 0.179 0.161 0.237 0.186

composition Hex2HexNAc2 Hex2HexNAc2Deoxyhexose1 Hex2HexNAc2Deoxyhexose2 Hex2HexNAc2NeuAc1 Hex3HexNAc2Deoxyhexose1 Hex3HexNAc3 Hex2HexNAc2Deoxyhexose3 Hex2HexNAc2Deoxyhexose1NeuAc1 Hex3HexNAc3Deoxyhexose1 Hex3HexNAc3Deoxyhexose2 Hex3HexNAc3NeuAc1 Hex4HexNAc3Deoxyhexose1 Hex4HexNAc4 Hex3HexNAc3Deoxyhexose3 Hex3HexNAc3Deoxyhexose1NeuAc1 Hex4HexNAc4Deoxyhexose1 Hex3HexNAc3Deoxyhexose2NeuAc1 Hex4HexNAc4Deoxyhexose2 Hex3HexNAc3Deoxyhexose3NeuAc1 Hex4HexNAc4Deoxyhexose3 Hex4HexNAc4Deoxyhexose1NeuAc1 Hex5HexNAc5Deoxyhexose1 Hex4HexNAc4Deoxyhexose4 Hex4HexNAc4Deoxyhexose2NeuAc1 Hex4HexNAc4NeuAc2 Hex5HexNAc5Deoxyhexose2 Hex4HexNAc4Deoxyhexose3NeuAc1 Hex5HexNAc5Deoxyhexose3 Hex5HexNAc5Deoxyhexose1NeuAc1 Hex6HexNAc6Deoxyhexose1 Hex5HexNAc5Deoxyhexose4 Hex6HexNAc6Deoxyhexose2 Hex6HexNAc6NeuAc1 Hex5HexNAc5Deoxyhexose3NeuAc1 Hex5HexNAc5Deoxyhexose1NeuAc2 Hex6HexNAc6Deoxyhexose3 Hex7HexNAc7Deoxyhexose1

O-glycans observed at m/z 879.3 (Figure 4a). The efficiency was considerably higher in the case of the other two structures observed at m/z 1240.4 and 1689.6 (Figure 4b,c, respectively). Profiling of O-Glycans Derived from a Highly Complex Glycoprotein. In order to demonstrate the potential of this technique to analyze real biological samples, O-glycans associated with bile salt-stimulated lipase (BSSL) isolated from human milk was cleaved following the enzymatic/chemical approach. BSSL has a molecular weight of 90-100 kDa and a carbohydrate content of approximately 20%.40 There is one possible site for N-glycosylation (N207) possessing complex-type glycans,41 while the carboxy-terminal region of BSSL is rich in serine and threonine residues, suggesting this domain to be highly O-glycosylated.42 It has been proposed that nine of these threonine residues in this region are occupied by O-linked oligosaccharides.43 As seen in Figure 5, the use of a 1 µg aliquot of BSSL purified from human milk was sufficient to identify more then thirty O-glycan compositions using the above-described enzymatic/chemical technique. Here, we observe a high level of both fucosylation and sialylation of the O-glycans in this glycoprotein, as consistent with (40) Landberg, E.; Huang, Y.; Stro ¨ mqvist, M.; Mechref, Y.; Hansson, L.; Lundblad, A.; Novotny, M. V.; Påhlsson, P. Arch. Biochem. Biophys. 2000, 377, 246–254. (41) Mechref, Y.; Chen, P.; Novotny, M. V. Glycobiology 1999, 9, 227–234. (42) Baba, T.; Downs, D.; Jackson, K. W.; Tang, J.; Wang, C.-S. Biochemistry 1991, 30, 500–510. (43) Wang, C.; Dashti, A.; Jackson, K. W.; Yeh, J.-C.; Cummings, R. D.; Tang, J. Biochemistry 1995, 34, 10639–10644.

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Table 3. Composition of Previously Unidentified BSSL O-Glycan Structures Shown in Figure 5 obs. mass calc. mass ∆mass 866.444 879.415 896.446 937.474 1083.522 1124.557 1130.570 1171.572 1212.616 1240.581 1298.649 1386.685 1519.819 1620.819 1661.817 1676.812 1689.819 1747.860 1818.850 1835.901 1921.966 1981.942 2009.975 2069.996 2098.040 2113.007 2156.022 2227.082 2268.065 2315.135 2472.168 2676.226 2762.295 3024.426 3299.491 3388.388 3473.497 3647.482

866.437 879.432 896.447 937.474 1083.532 1124.558 1130.558 1171.584 1212.611 1240.606 1298.647 1386.700 1519.762 1620.810 1661.837 1676.836 1689.832 1747.873 1818.911 1835.926 1921.963 1981.984 2010.015 2070.036 2098.031 2113.067 2156.073 2227.11 2268.137 2315.163 2472.236 2676.336 2762.373 3024.515 3299.652 3387.704 3473.741 3647.830

0.007 0.017 0.001 0 0.01 0.001 0.012 0.012 0.005 0.025 0.002 0.015 0.057 0.009 0.02 0.024 0.013 0.013 0.061 0.025 0.003 0.042 0.04 0.04 0.009 0.06 0.051 0.028 0.072 0.028 0.068 0.11 0.078 0.089 0.161 0.316 0.244 0.348

composition Hex1HexNAc1Deoxyhexose2 Hex1HexNAc1NeuAc1 Hex2HexNAc1Deoxyhexose1 Hex1HexNAc2Deoxyhexose1 Hex2HexNAc1NeuAc1 Hex1HexNAc2NeuAc1 Hex4HexNAc1 Hex3HexNAc2 Hex2HexNAc3 Hex1HexNAc1NeuAc2 Hex1HexNAc2Deoxyhexose1NeuAc1 Hex2HexNAc3Deoxyhexose1 Hex3HexNAc2Deoxyhexose2 Hex4HexNAc3 Hex3HexNAc4 Hex2HexNAc2Deoxyhexose2NeuAc1 Hex2HexNAc2NeuAc2 Hex2HexNAc3Deoxyhexose1NeuAc1 Hex2HexNAc4NeuAc1 Hex3HexNAc4Deoxyhexose1 Hex2HexNAc3Deoxyhexose2NeuAc1 Hex4HexNAc3NeuAc1 Hex3HexNAc4Deoxyhexose2 Hex5HexNAc4 Hex4HexNAc2NeuAc2 Hex3HexNAc3Deoxyhexose4 Hex4HexNAc3Deoxyhexose1NeuAc1 Hex4HexNAc4NeuAc1 Hex3HexNAc5NeuAc1 Hex5HexNAc5 Hex4HexNAc5NeuAc1 Hex5HexNAc5NeuAc1 Hex4HexNAc4Deoxyhexose1NeuAc2 Hex5HexNAc5Deoxyhexose2NeuAc1 Hex6HexNAc6Deoxyhexose1NeuAc1 Hex7HexNAc7Deoxyhexose1 Hex6HexNAc6Deoxyhexose2NeuAc1 Hex6HexNAc6Deoxyhexose3NeuAc1

our previously published data (Table 2).44 These structures were determined through a combination of MSMS (examples of which are illustrated in the Supporting Information) and enzymatic digestion. Moreover, there were additional structures that were not observed in our previous work (Table 3). This case demonstrates a high effectiveness of our new method, permitting the cleavage of large and complex O-glycans, originating from a glycoprotein that had been isolated and purified from a biological fluid (human milk). (44) Tegeler, T. J.; Mechref, Y.; Boraas, K.; Reilly, J. P.; Novotny, M. V. Anal. Chem. 2004, 76, 6698–6706.

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CONCLUSIONS The currently used methods for the analysis of O-linked oligosaccharides have numerous pitfalls. In the absence of a comprehensive enzymatic release for O-linked oligosaccharides, there has been a need to develop alternative cleavage methods which mimic enzymatic results. Previously, we have improved on the existing methods through the use of volatile reagents that minimized sample handling and, thus, improved sensitivity.31,32 Through this communication, we are further advancing the O-linked oligosaccharide cleavage through the use of a nonspecific proteolysis in conjunction with our solid-phase permethylation. This combination allows the release of free-reducing-end O-glycans which are simultaneously permethylated. The enzymatic/chemical method described here is efficient, repeatable, sensitive, and involves minimal sample handling. Moreover, the method does not substantially increase sample processing time in comparison to the other used procedures. This method also eliminates the use of several potentially hazardous chemicals, when compared to the existing β-elimination protocols. Altogether, the described approach furnishes about 100-fold improvement in sensitivity, allowing the analysis of O-glycans from low-microgram to submicrogram amounts of targeted glycoprotein samples. ACKNOWLEDGMENT This work was supported by grant No. GM24349 from the National Institute of General Medical Sciences, U.S. Department of Health and Human Services, and Grant No. RR018942 from the National Center for Research Resources, a component of the National Institute of Health (NIH-NCRR) for the National Center for Glycomics and Glycoproteomics (NCGG) at Indiana University. This work was also partially supported by the METACyt Biochemical Analysis Center, a component of the Indiana Metabolomics and Cytomics Initiative (METACyt), funded by a grant from Eli Lilly Endowment. The Authors also would like to acknowledge Prof. David R. Williams for his help understanding the mechanism of the cleavage. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 23, 2009. Accepted October 3, 2009. AC901363H