Mass Spectrometric Mapping and Sequencing of N-Linked

Anal. Chem. 1998, 70, 455-463

Mass Spectrometric Mapping and Sequencing of N-Linked Oligosaccharides Derived from Submicrogram Amounts of Glycoproteins Yehia Mechref and Milos V. Novotny*

Department of Chemistry, Indiana University, Bloomington, Indiana 47405

Very small quantities of glycoproteins were directly processed on a MALDI sampling plate prior to their mass spectrometric investigations. The on-plate digestion with N-glycanase released effectively the corresponding oligosaccharides in very short times, irrespective of their molecular mass. The following treatment with an array of exoglycosidase enzymes enables sequencing and a linkage-form determination in analysis times that are considerably shorter than achieved previously: the entire structural determination on a glycoprotein can be completed in one day, with a minimum substrate consumption. Ribonuclease B, bovine fetuin, human r1-acid glycoprotein, and the diamine oxidase (from porcine kidney) have been used to illustrate different aspects of the on-plate sample treatment/MALDI mass spectrometry.

Many biologically interesting proteins are glycosylated at their asparagine, serine, and threonine residues. Glycosylation has now been recognized as being more ubiquitous and structurally varied than all other types of posttranslational modifications combined. While glycoproteins are being increasingly implicated to be crucial to processes as diverse as cellular adhesion, egg fertilization, targeting aging cells, etc., very little is known about the underlying molecular basis of sugar-sugar and sugar-protein interactions. The extreme complexity of glycan structures, multiple substitutions (microheterogeneity) at glycosylation sites, and the structural diversity associated with the protein backbone itself thus represent an enormous task for analytical structural studies. Traditionally, a complete structural analysis of the glycan structures, including determination of the carbohydrate sequence and sugar linkage forms, has been a tedious, multitechnique task, often necessitating milligram to gram quantities of material. To progress from this situation, it is first essential to have a sensitive end-measurement methodology. The recent advances in matrixassisted laser desorption/ionization(MALDI) mass spectrometry (MS) have provided very sensitive means for the analysis of oligosaccharides.1-5 During the recent period, this gain in sensitivity has enabled several research groups to use MS in * Corresponding author: (phone) 812-855-4532; (fax) 812-855-8300; (e-mail) [email protected]. (1) Harvey, D. J. J. Chromatogr., A 1996, 720, 429-446. (2) Harvey, D. J.; Rudd, P. M.; Bateman, R. H.; Bordoli, R. S.; Howes, J. B.; Vickers, R. G. Org. Mass Spectrom. 1994, 29, 753-765. S0003-2700(97)00947-5 CCC: $15.00 Published on Web 02/01/1998

© 1998 American Chemical Society

conjunction with selective exoglycosidase cleavages for the characterization of N-glycans.6-10 Since direct observation of glycans in intact glycoproteins is currently not feasible, it is also important that the methods for glycan release and further purification do not negate the obtainable sensitivity of the final MS determination. When working with minute quantities of glycoproteins, severe sample losses during their manipulation can easily become a bottleneck in such determinations. Thus, a minimum number of sample manipulation steps appears desirable in extending the state-of-the-art methodologies to the submicrogram area. The use of enzyme reagents is rapidly becoming popular in modern carbohydrate analysis because of their inherent selectivities for a sugar substrate and its linkage type. In addition to the availability of different exoglycosidases (for the benefit of enzymatic sequencing), various glycanases to cleave particular oligosaccharides from a polypeptide backbone are also being increasingly utilized. Traditionally, a problem with such enzymatic digestion has been the relatively long times; for their reactions in solutions, many hours are typically needed. In this report, we demonstrate new capabilities of performing the enzymatic cleavages (associated with both the glycan release and the sequencing enzyme arrays) directly on a typical MALDI sampling plate. It will be shown that such enzymatic treatments, when carried out in small volumes and surface areas, can yield analytically sufficient quantities of glycans for the display of oligosaccharides and subsequent determination of sequence and the linkage forms through MALDI/MS. The enzymatic reactions proceed at very fast rates, leading often to the acquisition of needed structural information in one day or less. Because of avoiding the usual sample transfers and dilution steps through the on-plate procedure, the sensitivity potential of MALDI/MS is fully utilized. As demonstrated with the example of several glycoproteins, complete structural information is feasible from the low-microgram and submicrogram quantities of a glycoprotein (3) Karas, M.; Ehring, H.; Nordhoff, E.; Stahl, B.; Strupat, K.; Hillenkamp, F.; Grehl, M.; Krebs, B. Org. Mass Spectrom. 1993, 28, 1476-1481. (4) Papac, D. I.; Wong, A.; Jones, A. J. S. Anal. Chem. 1996, 68, 3215-3223. (5) Rouse, J. C.; Vath, J. E. Anal. Biochem. 1996, 238, 82-92. (6) Sutton, C. W.; O’Neill, J. A.; Cottrell, J. S. Anal. Biochem. 1994, 218, 3446. (7) Yang, Y., Orlando, R., Anal. Chem. 1996, 68, 570-572. (8) Mortz, E.; Sareneva, T.; Julkunen, I.; Roepstorff, P. J. Mass Spectrom. 1966, 31, 1109-1118. (9) Harmon, B. J.; Gu, X.; Wang, D. I. C. Anal. Chem. 1966, 68, 1465-1473. (10) Zhao, Y.; Kent, S. B. H.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1629-1633.

Analytical Chemistry, Vol. 70, No. 3, February 1, 1998 455

Figure 1. MALDI mass spectrum of ribonuclease B before (a) and after treatment (b) with PNGase F. The inset represents the profile of N-glycans after digestion of 5 µg of ribonuclease B for 1 h on the plate.

placed on the MALDI plate. Although, at this stage, we demonstrate this capability for the N-linked oligosaccharides only, this approach may place the field of glycoprotein analysis closer to some of the most challenging biomolecules (e.g., receptor proteins at biological membranes). EXPERIMENTAL SECTION Materials. The glycoproteins (ribonuclease B, human R1-acid glycoprotein (AGP), bovine fetuin, diamino oxidase from porcine kidney (DAO) and ovalbumin) were obtained from Sigma Chemical Co. (St. Louis, MO). The other enzymes and reagents obtained from Sigma included 2,5-dihydroxybenzoic acid (DHB) and neuraminidase (EC 3.2.1.18) from Arthrobacter ureafaciens. N-glycosidase F (PNGase F) of Flavobacterium meningosepticum, a recombinant form from Escherichia coli (EC 3.2.2.18), N-acetylβ-D-glucosaminidase from Diplococcus pneumoniae (EC 3.2.1.30), and β-galactosidase from D. pneumoniae (EC 3.2.1.23) were from Boehringer Mannheim Corp. (Indianapolis, IN). The remaining chemicals used in this study were purchased from Aldrich (Milwaukee, WI). Methods. Enzymatic Cleavages. Enzymatic release and sequencing of N-glycans were performed on the MALDI sample plate according to the following procedure. Low-microgram and 456

Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

submicrogram quantities of glycoprotein to be analyzed were deposited on the plate and reconstituted in 1 µL of sodium phosphate buffer (10 mM concentration, pH 6.5). Typically, 30 munits of PNGase F were added to each spot. For enzymatic sequencing, three glycoprotein spots were deposited on the MALDI plate and different enzyme arrays were added to each spot. PNGase F (30 munit) and neuraminidase (3 munit) were added to the first spot. PNGase F, neuraminidase and β-galactosidase (0.3 munit) each were added to the second spot, while PNGase F, neuraminidase, β-galactosidase, and N-acetyl-β-Dglucosaminidase (0.3 munit each) were added to the third spot. The plate was then placed in a crystallization beaker containing water on the bottom. The beaker was covered with Parafilm and placed in a water bath set to 40 °C for 3 h. High-purity water obtained from Stephens Scientific (Riverdale, NJ) was continuously added to the spots to prevent drying. While all enzymatic releases of N-glycans and their subsequent enzymatic sequencing were performed on the MALDI plate, the N-glycans from ovalbumin were in parallel enzymatically released in microtubes for a comparison. Briefly, 1 mg of ovalbumin to be digested was reconstituted in 10 mM sodium phosphate buffer (pH 7.5), followed by addition of PNGase F (5 munits/0.1 mg of glycoprotein). The reaction mixture was subsequently incubated

Figure 2. MALDI mass spectra representing the N-linked oligosaccharide profiles derived from ovalbumin by digestion overnight (a), digestion of 5 µg for 3 h on the plate (b), and digestion of 5 µg on the plate, after thermal denaturation of the glycoprotein (c).

for 18 h at 37 ˚C. The enzymatically released oligosaccharides were recovered by applying the reaction mixtures to a C18 SepPak cartridge (Waters Corp., Milford, MA) that was preconditioned with methanol, acetonitrile, and aqueous methanol (1:19 v/v). The oligosaccharides were eluted with 3 mL of 10% aqueous methanol, lyophilized, and reconstituted in water to a glycoprotein initial concentration of 2 mg/mL. A MALDI/MS analysis of this sample was performed with a 1-µL aliquot. Instrumentation. All MALDI mass spectra were acquired on a Voyager-DE RP Biospectrometry workstation (PerSeptive Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser (337 nm). The instrument can operate in both the reflector and linear modes, but the linear mode was used exclusively in this study. The MALDI spectra were acquired at 25-kV accelerating voltage, with delay extraction, which was set to 150 ns. The instrument was externally calibrated with the maltose ladder, encompassing the m/z values of the analyzed sample constituents (two-point calibration). All acquired spectra were smoothed by applying the 19-point Savitzky-Golay smoothing.11 The MALDI instrument used in this report utilizes a multichannel plate detector, and in order to preclude the low-mass ions from saturating the detector, the low-mass ion gate was set at 800 Da. (11) Savitzky, A.; Golay, M. J. E. Anal. Chem. 1964, 36, 1627-1639.

For acquiring the MALDI spectra of the cleaved or sequenced N-glycans, a recently reported arabinosazone matrix was utilized,12 while DHB served as the MALDI matrix for the intact ribonuclease B. The arabinosazone matrix was prepared in ethanol at 10 mg/ mL concentration. The DHB matrix was prepared as a saturated solution in ethanol. Sample spots were allowed to dry first, before 1.0-µL matrix solutions were added. RESULTS AND DISCUSSION Traditionally, a complete structural analysis of glycoproteins represents a tedious, long-term task involving the solution cleavages of glycans from the polypeptide backbone, separation and purification steps, and, finally, spectroscopic determination of the individual glycans. Besides the obvious procedural complexity, sample losses necessarily occur during the multiple manipulations. While the advent of MALDI/MS and its ancillary techniques represents a major step forward in terms of sensitive detection and structural analysis, there is an obvious mismatch between its capabilities and the “wet chemistry” steps preceding the final measurement. Processing of a purified glycoprotein on the MALDI sample plate is an attractive approach that can overcome at least some of (12) Chen, P.; Baker, A. G.; Novotny, M. V. Anal. Biochem. 1997, 244, 144151.


457

the above mentioned difficulties. Through the structural analysis of selected N-linked oligosaccharides, we wish to demonstrate here the power of this approach. As shown below on several examples, the enzymatic cleavage of N-glycans, profiling of the cleaved oligosaccharides, and enzyme-based sequencing and linkage determination can be accomplished on the sample plate without sample removal or any other manipulation which could result in a sample loss. The entire analytical procedure is very fast and sensitive. As a direct consequence of the Michaelis-Menten theory relating the initial velocity of an enzymatic reaction to substrate concentration, conducting the enzymatic reactions in a small volume allows the substrate concentration to approach Km (Michaelis constant) and thus decrease the incubation time needed for completion. This advantage was pointed out by Ku¨ster et al.13 in relation to sequencing of oligosaccharides through the action of exoglycosidases. However, in their procedure, tedious recovery and sample reconstitution steps were involved to complete structural investigation. They also determined that high buffer concentration (as commonly used for enzymatic reactions in solutions) was not necessary for an effective exoglycosidase digestion. In agreement with this finding,13 we ran all our onplate reactions in 10 mM phosphate buffer. In the work presented here, all enzymatic cleavages and a subsequent enzymatic sequencing were carried out exclusively on the plate (without a recovery), necessitating only lowmicrogram (and often less than microgram) quantities of the studied glycoproteins. A digestion with N-glycanase would be deemed a most challenging step because of the large size of glycoprotein substrates. (An enzymatic cleavage with PNGase F in the conventional test tube arrangement is known to take many hours.) As shown with the specific examples below, this type of enzymatic cleavage can be accomplished on the plate in a relatively short time, and additionally, a glycoprotein’s molecular weight has little effect on the success of this procedure. This is demonstrated with several glycoproteins which range from 14 to 105 kDa of molecular mass. This is, presumably, due to a facile conversion of these substrates in a small volume and/or the surface effects. The initial trials to cleave oligosaccharides through the action of PNGase F were conducted with a small glycoprotein, ribonuclease B. The MALDI spectrum of this glycoprotein is illustrated in Figure 1a. Different glycoforms are known to exist in this glycoprotein, which has one site available for N-glycosylation.14 Singly and doubly charged glycoprotein forms are observed in the spectrum. Singly and doubly charged ions will also be observable in ribonuclease A (deglycosylated form of ribonuclease B). It turned out that the enzymatic cleavage of the oligosaccharides was completed after just 1 h of incubation, as was determined from the MALDI analysis of the samples incubated on the plate with PNGase F for different lengths of time (Figure 1b). Note that no traces of an incomplete or partial digestion of the ribonuclease were observed in the spectrum after just a 1-h digestion. The N-linked oligosaccharide profile of ribonuclease B is depicted in the inset of Figure 1b in which the five oligosaccharides that are known to exist2,14 in ribonuclease B were (13) Ku ¨ ster, B.; Naven, T. J. P.; Harvey, D. J. J. Mass Spectrom. 1996, 31, 11311140. (14) Fu, D.; Chen, L.; O’Neill, R. A. Carbohydr. Res. 1994, 261, 173-186.

458 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

Table 1. Structures of the N-Glycans Cleaved from Ovalbumin [M + Na]+ exp

obs

933.80

933.85

1137.00

1137.19

1258.09

1258.36

1340.19

1340.25

1420.23

1420.46

1461.30

1461.49

1502.32

1502.44

1543.38

1543.44

1664.47

1664.47

1705.52

1705.46

1746.57

1746.62

1867.66

1867.76

1908.70

1908.67

1949.76

1949.79

2153.0

2153.35

structuresa

a Obtained from refs 13 and 22-24: 9, N-acetylglucosamine; O, mannose; 0, galactose; 3, fucose.

readily observed. The mismatch between the observed molecular ions of the glycoforms displayed in the figure inset (accuracy better than 0.1%) and the mass difference between the parent protein and ribonuclease A was found to be due to formation of the sodium adducts in the very concentrated buffer solution. The broad peak observed for ribonuclease A was also due to sodium adduction. (Washing salts from the sample spot narrowed the peak considerably and returned the correct mass.) Obviously, digestion of this simple glycoprotein on the plate was feasible with PNGase, encouraging studies of more challenging glycoproteins.

Table 2. Endo- and Exoglycosidase Specificity and Optimum pH

f

enzyme

specificity

opt pHa

N-glycosidase F, recombinant (PNGase F) β-galactosidase from D. pneumoniae N-acetyl-β-D-glucosaminidase from D. pneumoniae neuraminidase from A. ureafaciens

cleaves high-mannose structures, hybride structures, and complex structures hydrolyzes terminal galactose residues that are β(1-4)-linked to GlcNAcb cleaves terminal GlcNAc residues that are β(1-2)-linked to Manc cleaves terminal sialic acid residues that are R(2-3)-, R(2-6)-, or R(2-8)-linked to Gal,d GlcNAc, GalNAc,e and AcNeu,f GlcNeug residues

5.0-7.0 6.0-6.5 5.0 5.0-5.5

a Values obtained from Boehringer Mannheim Biochemicals Catalog. b N-Acetylglucosamine. c Mannose. d Galactose. e N-Acetylgalactosamine. N-Acetylneuraminic acid. g N-Glycoloylneuraminic acid.

Figure 3. MALDI mass spectra of the N-linked oligosaccharide profile recorded from 5 µg of bovine fetuin after treatment with PNGase F and neuraminidase (a); PNGase F, neuraminidase, and galactosidase (b); and PNGase F, neuraminidase, galactosidase, and N-acetylglucosaminidase (c); all at pH 7.5. The inset represents the m/z range where the sialylated species appear as multiple sodium adducts. The peaks marked with an arrow are contaminants originating from the enzyme preparations.

Note that the ability to use low-concentration buffer as well as a neutral matrix prepared in neat organic solvents reduced salt interference and resulted in strong MALDI signals corresponding to the oligosaccharide structures. In addition, it is critical to maintain the sample spot with the enzyme in a moist condition during digestion. Ovalbumin was subsequently chosen for demonstration of our procedure with a very complex glycoprotein. The oligosaccharides of this glycoproteins have been structurally characterized through different methodologies, with numerous reports on their structures now available.15-20 Although ovalbumin, like ribonu-

clease B, has a single site available for glycosylation, there are known to be more than 20 oligosaccharide structures in existence. The average molecular weight of ovalbumin, excluding the mass (15) Tai, T.; Yamashita, K.; Ogata-Arakawa, M.; Koide, N.; Muramatsu, T.; Iwashita, S.; Inoue, Y.; Kobata, A. J. Biol. Chem. 1975, 250, 8569-8575. (16) Tai, T.; Yamashita, K.; Ito, S.; Kobata, A. J. Biol. Chem. 1977, 252, 66876694. (17) Yamashita, K.; Tachibana, Y.; Kobata, A. J. Biol. Chem. 1978, 253, 38623869. (18) Chen, L.-M.; Yet, M.-G.; Shao, M.-C. FASEB J. 1988, 2, 2819-2824. (19) Yet, M.-G.; Chin, C. C. Q.; Wold, F. J. Biol. Chem. 1988, 263, 111-117. (20) Maley, F.; Trimble, R. B.; Tarentino, A. L.; Plummer, T. H. Anal. Biochem. 1989, 180, 195-204.


459

Figure 4. MALDI mass spectra of the N-linked oligosaccharide profile derived from 5 µg of bovine fetuin after treatment with PNGase F and neuraminidase (a); PNGase F, neuraminidase, and galactosidase (b); and PNGase F, neuraminidase, galactosidase, and N-acetylglucosaminidase (c); all at pH 6.5. The inset represents the m/z range where the sialylated species are expected to appear as multiple sodium adducts. The peaks marked with an arrow are contaminants originating from the enzyme preparations.

of the N-linked glycans, is 42 869.21 The ability to completely digest a minute amount of this glycoprotein on the plate should undoubtedly illustrate the effectiveness of our approach in the analysis of various glycoforms existing in a glycoprotein regardless of its complexity. The MALDI spectra of N-linked oligosaccharides cleaved from ovalbumin, under different experimental conditions, are depicted in Figure 2. The MALDI spectrum of the oligosaccharides cleaved by a solution digestion for 18 h in a microtube resembles remarkably the spectrum observed after the glycoprotein was digested on the plate for 3 h (Figure 2a vs b), while the relative intensities of their signals are almost identical. However, the high-mannose structures that are known to exist in ovalbumin22-24 were not observed in either sample. Denaturation of the protein by boiling the sample for 10 min prior to the (21) Duffin, K. L.; Welply, J. K.; Huang, E.; Henion, J. D. Anal. Chem. 1992, 64, 1440-1448. (22) Corradi Da Silva, M. L.; Stubbs, H. J.; Tamura, T.; Rice, K. G. Arch. Biochem. Biophys. 1995, 318, 465-475. (23) Naven, T. J. P.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1996, 10, 1361-1366. (24) Honda, S.; Makino, A.; Suzuki, S.; Kakehi, K. Anal. Biochem. 1990, 191, 228-234.


enzymatic cleavage not only improved the intensity of all signals observed but also allowed determination of the high-mannose structures that were not observed in the absence of the denaturation step (Figure 2c, with structures listed in Table 1). Denaturation of high-molecular-weight glycoprotein appears desirable for a complete determination of the structures existing in a particular biomolecule. Thus far, we have shown the effectiveness of an on-plate enzymatic cleavage of oligosaccharides through the action of PNGase F for two glycoproteins with a different heterogeneity yet the same number of glycosylation sites. The next goal of this study was to perform a simultaneous cleavage and sequencing of the oligosaccharide structures, all on the sample plate. Such an approach should eliminate unnecessary sample handling and the losses that are usually encountered in sample workup. The enzymatic cleavages and sequencing have been performed simultaneously on the plate by depositing several spots containing the different array of enzymes needed for the enzymatic sequencing, as explained in the Experimental Section. In this study, three exoglycosidases were utilized to enzymatically sequence oligosaccharide structures: neuraminidase from A. ureafaciens, N-acetyl-β-D-glucosaminidase from D. pneumoniae, and

Figure 5. MALDI mass spectra of the N-linked oligosaccharide profile derived from 100 ng of bovine fetuin after treatment with PNGase F and neuraminidase (a); PNGase F, neuraminidase, and galactosidase (b); and PNGase F, neuraminidase, galactosidase, and N-acetylglucosaminidase (c); all at pH 6.5. The peaks marked with an arrow are contaminants originating from the enzyme preparations.

β-galactosidase from D. pneumoniae. These enzymes were added to the spots in addition to the PNGase F enzyme that had been applied to all spots earlier. The fact that these exoglycosidases have different pH optimums has prompted the investigation of a compromise pH for all. The optimum pH values for the enzymes are summarized in Table 2. This investigation was performed on bovine fetuin which is an R-globulin with an average molecular mass of 48 kDa and has often been used as a model for the study of glycoprotein structure and biosynthesis.25 Fetuin possesses six carbohydrate structures per molecule, three are O-glycosidically linked to Ser/Thr26 and three N-glycosidically linked to Asn.27-30 Moreover, fetuin emerged as a likely candidate for evaluating the effectiveness of this procedure because of the presence of several structures that feature the same m/z values yet are different in the linkage (β(1-4) vs β(1-3)) of galactose residues. (25) Green, E. D.; Adelt, G.; Baenziger, J. E.; Wilson, S.; Van Halbeek, H. J. Biol. Chem. 1988, 263, 18253-18268. (26) Spiro, R. G.; Bhoyroo, V. D. J. Biol. Chem. 1974, 249, 5704-5717. (27) Spiro, R. G. J. Biol. Chem. 1963, 238, 644-649. (28) Spiro, R. G. J. Biol. Chem. 1960, 235, 2860-2869. (29) Spiro, R. G. J. Biol. Chem. 1962, 237, 382-388. (30) Spiro, R. G. J. Biol. Chem. 1962, 237, 646-652.

Initially, the enzymatic sequencing was attempted in phosphate buffer at pH 7.5 which is the optimum pH for PNGase F activity. Although it appeared that neuraminidase effectively cleaved some terminal sialic acid structures at this pH, the signal observed in the m/z range corresponding to the sialylated structures proved these cleavages to be incomplete (inset in Figure 3a). While the galactosidase cleaved completely the terminal galactose residues that are β(1-4)-linked (Figure 3b), N-acetyl-β-D-glucosaminidase partially hydrolyzed terminal GlcNAc residues that are β(1-2)linked to the mannose residue (Figure 3c). Consequently, the pH for the enzymatic reaction was lowered to 6.5 in order to improve the activity of neuraminidase and N-acetyl-β-D-glucosaminidase. Lowering the pH resulted in a high neuraminidase activity, as was evident by the absence of any signals corresponding to sialylated structures (Figure 4a and its inset). Moreover, lowering the pH to 6.5 also resulted in a higher activity for N-acetyl-β-Dglucosaminidase and a complete cleavage of the terminal GlcNAc residues that are linked to mannose through β(1-2) linkage (Figure 4c). This was evident from the observation of only three m/z signals at pH 6.5, as compared to eight m/z signals at pH 7.5. The signal observed at m/z 1299 in Figure 3c corresponds to a sodium adduct of the oligosaccharide structure that has a Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

461

Figure 6. MALDI mass spectra of N-linked oligosaccharide profile derived from 5 µg of AGP after treatment with PNGase F and neuraminidase (a); PNGase F, neuraminidase, and galactosidase (b); and PNGase F, neuraminidase, galactosidase, and N-acetylglucosaminidase (c). The peaks marked with an arrow are contaminants originating from the enzyme preparations.

Gal-β(1-3)GlcNAc residue on one of the antennas, corresponding to a type of linkage observed previously in fetuin oligosaccharides.25 The signals observed at m/z values of 1388, 1408, and 1442 (in Figures 3b and 4b) are contaminants in the exoglycosidase preparations. Lowering the pH further to 6.0 had no effect on the observed MALDI spectra (data not shown). Moreover, the procedure was successfully performed on 100 ng of the glycoprotein and the full structures were determined by utilizing only 300 ng of this glycoprotein (Figure 5). The on-plate sequencing approach was also tested on AGP, a glycoprotein with an average molecular mass of 52 kDa. The three oligosaccharide structures that are known to be N-linked in AGP31,32 were seen after treatment of the glycoprotein with PNGase F and neuraminidase (Figure 6a). A sequential enzymatic digestion of AGP verified the structures and linkages of its saccharides (b and c) which agree with that reported in the literature.31,32 Once again, this case demonstrates how effective (31) Fournet, B.; Montreuil, J.; Strecker, G.; Dorland, L.; Haverkamp, J.; Vliegenthart, F. G.; Binette, J. P.; Schmid, K. Biochemistry 1978, 17, 52065214. (32) Yoshima, H.; Matsumoto, A.; Mizuochi, T.; Kawasaki, T.; Kobata, A. J. Biol. Chem. 1981, 256, 8476-8484.


the entire on-plate procedure is, beginning with the glycoprotein digestion and terminating with the determination of sequence and linkage forms. The signals that sometimes originate from the enzyme contamination are very weak and easily identified through running a blank. The entire structure determination in the case of AGP was derived from just 15 µg of this glycoprotein. The peaks were judged to be meaningful signals when the signal-tonoise ratio was more than 3. The last glycoprotein tested in this work was diamine oxidase from porcine kidney which has 105-kDa molecular mass and contains only 10% N-linked oligosaccharides. The complete structure was recently elucidated in our laboratory33 through a combination of the conventional (solution) enzymatic digestion procedures and MALDI tandem mass spectrometry (MS/MS) techniques. While milligram quantities of DAO were earlier required to complete structural determination, an equivalent amount of information was gathered through the on-plate technique with just 15 µg of this glycoprotein. A problem encountered during the investigation of this glycoprotein enzyme was contamination of the DAO preparation with poly(ethylene glycol) (33) Huang, Y.; Mechref, Y.; Novotny, M., in preparation, 1997.

Figure 7. MALDI mass spectra of the N-linked oligosaccharides derived from 5 µg DAO after treatment with PNGase F (a); PNGase F and neuraminidase (b); PNGase F, neuraminidase, and galactosidase (c); and PNGase F, neuraminidase, galactosidase, and N-acetylglucosaminidase (d). The peaks marked with an arrow are contaminants from the enzyme preparations and the sample itself (presumably, poly(ethylene glycol) used as a stabilizer).

Table 3. Structures of the N-Glycans Derived from DAO [M + Na]+

structures

[M + Na]+

918.70

1665.69

1284.25

1811.83

1341.31

1974.32

1446.23

2138.37

1487.40

2177.20

1503.92

2543.06

structures

1649.65

(presumably, used as a stabilizer). In spite of this drawback, the digestion and a subsequent enzymatic sequencing proceeded smoothly. The contamination peaks, appearing in the same general area as the oligosaccharide peaks (Figure 7), could be sorted out from the structurally significant signals. Short-hand

structures for the N-glycans determined in Figure 7 are summarized in Table 3. CONCLUSIONS Using several glycoproteins as examples, we have repeatedly verified that digestions with N-glycanase and the mixtures of specific exoglycosidases can all be carried out directly on a MALDI sample plate, leading to a total structural characterization of N-glycans in such glycoproteins. Among the most important advantages of this approach is avoidance of sample losses which are typically encountered in a more conventional glycoanalysis. Sequencing and linkage analysis can be performed in relatively short times. Further improvements in MALDI/MS instrumentation and sample matrixes are likely to lead to further sensitivity improvements. Developing parallel capabilities for O-linked glycans becomes increasingly important. ACKNOWLEDGMENT This study was supported by Grant GM24349 from the National Institute of General Medical Sciences, U.S. Department of Health and Human Services. We thank Andrew G. Baker for his technical assistance. Received for review August 26, 1997. Accepted November 21, 1997.X AC970947S X

Abstract published in Advance ACS Abstracts, December 15, 1997.


463

Mass Spectrometric Mapping and Sequencing of N-Linked

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