Gas chromatography and mass spectrometry of disaccharides from glycoproteins

Journal of Biochemical and Biophysical Methods, 1 (1979) 227--235 © Elsevier/North-Holland Biomedical Press

227

GAS CHROMATOGRAPHY A N D MASS SPECTROMETRY OF DISACCHARIDES FROM GLYCOPROTEINS

C. ALLEN BUSH

Department of Chemistry, Illinois Institute of Technology, Chicago, IL 60616, U.S.A. (Received 4 January 1979; accepted 15 May 1979)

Partial acid hydrolysis and methanolysis released disaccharides and disaccharide methylglycosides from the glycoproteins, ovomucoid and porcine gastric mucin in amounts of 0.5--7 /lg disaccharide per mg of glycoprotein. These disaccharides were fractionated by gas chromatography as the trimethylsilyl (Me3Si) derivatives. The composition of recovered disaccharides has been determined by hydrolysis and rechromatography of the Me3Si monosaccharides. The intersaccharide linkages of the disaccharides have been determined by electron impact mass spectrometry. This simple and rapid method can give structural information on small glycoprotein samples. Key words: methanolysis; hydrolysis; silylation; glycoproteins; gas chromatography ; mass spectrometry.

INTRODUCTION Determination of the complete covalent chemical structure of the oligosaccharide chains of glycoproteins is a difficult task generally requiring large samples. The linkage positions and carbohydrate sequence are often elucidated by methylation analysis or by periodate degradation. Specific exoglyeosidases have been used to determine sequence and anomeric configuration but their use generally gives no information on linkage position. A less widely used approach to oligosaccharide structural analysis is that of isolating disaecharides, trisaccharides, etc., by chemical degradation of a glycoprotein. For a typical asparagine-linked glycoprotein (ovomueoid), Bayard et al. [I] have isolated six different disaccharides and six larger oligosaecharides using hydrolysis with acidic resin (Dowex 50). These workers have isolated milligram quantities of each disaccharide and oligosaccharide in order to carry out structure determination using classical methods. Starting with I g of hydrolyzate, they fractionate oligosaccharides by size on charcoal C°lumns and purify them by preparative paper chromatography on 3 MM paper [I]. This method could become considerably more attractive as a method for deriving information on glycoprotein structure if a rapid and i Abbreviations: GC, gas chromatography; EI-MS, electron impact mass spectrometry.

228

sensitive m e t h o d for separating oligosaceharides and determining their structure were available. Several laboratories have reported suceessful fractionation of oligosaceharides by gas chromatography (GC) of the MeaSi derivatives [21 as well as correlations of electron impact mass spectra (EI-MS) with glycosidic linkage [3,4]. In this paper preparation of several disaecharides by partial hydrolysis and methanolysis of ovomueoid and porcine gastric mucin is reported. Further, their fractionation on a small scale by GC as the Me3Si derivatives and methods for structure determination using mass spectrometric techniques are described. EXPERIMENTAL

Ovomucoid and porcine gastric mucin from Sigma were used without further purification. Acidic resin served as catalyst for glyeoprotein hydrolysis. 10 mg of ovomucoid were dissolved in 1.5 ml of water and suspended with 1.5 ml of washed Bio-Rad AG-50 WX8 (H+). The mixture was incubated for varying periods in capped tubes at 100 or 80°C, cooled, and the resin filtered off and rinsed. In some experiments protein was separated from released sugars by filtration in an Amicon high-pressure dialysis cell using a PM-10 membrane. In our procedure for aeidie methanolysis, 10 mg of glyeoprotein was incubated in 3 ml of 0.5 N anhydrous methanolic HC1. Chloride Was precipitated by adding silver carbonate until the solution was neutral. The clear solution resulting from the hydrolysis was freeze dried and that from methanolysis evaporated, and the resulting residue was silylated. Consistent quantitative silylation of impure mixtures of disaceharides was f o u n d using the procedure of Brobst and Lott [5]. The residue was dissolved in 20 gl of pyridine at 70°C in a Reacti-Vial (Pierce). After cooling, 20 #1 of hexamethyldisilazane were added followed by 2 pl of trifluoroaeetie acid and the mixture was heated at 70°C for 30 mino Gas chromatography was carried out with a Varian model 2445 with flame ionization detector on columns of 3% SE-30 on Chrom W H/p with nitrogen as the carrier gas. Monosaecharides fraetionate conveniently at 160°C and disaccharides separate well at 225 ° C. For the purpose of identification, the disaccharides were collected with an effluent splitter which diverted part of the sample gas away from the flame detector where it could be collected in a Pasteur pipet [6], In order to determine its m o n o m e r composition, the Me3Si disaccharide (1--10 pg) was collected in a Pasteur pipet and rinsed with benzene into a Reaeti-Vial; 250 #1 of 2 N H2SO4 was then added and the mixture was incubated for 2 h at 100°C, After cooling, the acid was neutralized with barium carbonate, and the supernatant drawn off and freeze dried for subsequent analysis for monosaccharides. Me3Si reagent was added to the dry residue and the mix2 ture chromatographed at 160 ° C. Mass spectra were measured with a Varian MAT CH-7 instrument using

229 either a direct probe inlet when the GC effluent was collected, or by directly interfaced GC--MS using a silicone membrane interface maintained at 220 ° C. Similar mass spectra for Me3Si disaccharides were observed by the two methods.

RESULTS GC at 1 6 0 ° C o f t h e h y d r o l y z a t e o f o v o m u c o i d s h o w e d peaks w h o s e retent i o n times and a n o m e r i c ratios c o r r e s p o n d to t h o s e o f a q u e o u s equilibrium m i x t u r e s o f m o n o s a c c h a r i d e s . T h e relative r e t e n t i o n times were in g o o d a g r e e m e n t with t h o s e r e p o r t e d b y Bhatti et al. [ 6 ] . C o m p a r i s o n o f t h e p e a k heights in t h e o v o m u c o i d h y d r o l y z a t e with t h o s e f r o m m o n o s a c c h a r i d e standards allowed estimates o f m o n o s a c c h a r i d e s released as f u n c t i o n o f h y d r o l y s i s time. The results in Table 1 are in a g r e e m e n t with t h o s e f o u n d b y M o n t r e u i l et al. [7] using p a p e r c h r o m a t o g r a p h i c analysis. G l c N A c a n d galactose are released u n d e r mild h y d r o l y t i c c o n d i t i o n s while release o f m a n n o s e requires m o r e extensive h y d r o l y s i s . T h e decrease in G l c N A c at long h y d r o l y s i s times is due to d e - N - a c e t y l a t i o n a n d binding t o t h e resin. T h e Me3Si derivatives o f t h e m o n o s a c c h a r i d e m e t h y l g l y c o s i d e s resulting f r o m partial m e t h a n o l y s i s o f o v o m u c o i d gave gas c h r o m a t o g r a m s quite similar to those o b s e r v e d in the c a r b o h y d r a t e analysis m e t h o d o f C l a m p et al. [8]. As in t h e case o f h y d r o l y s i s , the time course o f m o n o s a c c h a r i d e release b y m e t h a n o l y s i s o f o v o m u c o i d (Table 2) shows early release o f

TABLE

1

YIELDS OF THE Me3Si PRODUCTS WITH ACIDIC RESIN AG-50-W

OF PARTIAL

HYDROLYSIS

OF OVOMUCOID

Gas chromatography on 3% SE-30,225 ° C. Retention times relative to (~-lactose. Yields in pg of carbohydrate per mg of glycoprotein. Rel. retention time Monosaccarides Mannose Galactose GlcNAc Disaccharides A B

I.i0 1.20

B2

1.41

C D

1.61 2.00

Hydrolysis time at 80 ° C 90 rain

3h

3.4 3 14

5 4 17

0.8 0.6 0.1 0.i 0.5

1.0 0.5 0.1 0.3 0.5

5h

8 4 16 1.0 1.0 0.3 0.5 0.5

Hydrolysis time at 100 ° C 16 h

8 6 20 1.0 1.4 0.3 0.6 0.5

20 min

40 rain

90 min

17 11 23

20 12 12

28 20 6

0.5 2.0 0.8 0.7 0.4

1.0 2.0 0.6 0.8 0.4

1.0 3.0 2.0 1.5 0.6

230 TABLE

2

YIELDS OF THE Me3Si COID WITH 0.5 N HC]

PRODUCTS

OF

PARTIAL

METHANOLYSIS

OF

OVOMU-

Gas chromatography on 3% SE-30,225°C for disaccharides. Retention times for disaccharides relative to S-lactose. Yields in ~g carbohydrate per mg of protein. Rel. retention

Methanolysis time at 65 ° C 20 min

ih

Methanolysis time at 80 ° C

2 h

time Monosaccharides MannQse Galactose GicNAc Disaccharides

1.12

3 9 48 0.3

14 16 60 1.6

1.75

2

0.7

36 27 80 5

0.3

20 m i n

1 h

53 16 91 7

70 30 100 1.5

--

--

GIcNAc and galactose, with mannose appearing later. Carbohydrate analysis of this ovomucoid sample by the method of Clamp et al. [8] gives 3% (w/w) galactose, 8% mannose and 22% GlcNAco Since porcine gastric mucin is not completely water soluble~ it is not amenable to hydrolysis with the insoluble resin, Dowex 50. Partial methanolysis leads to monosaccharide release, as indicated in Table 3. In contrast to the case of ovomucoid, the constituent sugars are released more uniformly from porcine gastric mucin. Gas chromatography at 225°C of the ovomucoid hydrolyzate revealed as

TABLE

3

YIELDS OF THE Me3Si PRODUCTS OF PARTIAL METHANOLYSIS OF PORCINE GASTRIC MUCIN WITH 0.5 N HCI Gas chromatography on 3% SE-30,225°C for disaccharides. Retention times for disaccharides are relative t o ~ q a c t o s e . Yields in ~g c a r b o h y d r a t e per m g p r o t e i n . Rei. retention time Monosaccharides Mannose Galactose GlcNAc GalNAc NANA Disaccharides

1.02 1.53 1.76

65 ° C, 20 m i n

<30 15 9 13 5 0.3 3 6

65 ° C, 1 h

<30 57 42 31 10 1 5 5

8 0 ° C, 1 h

<39 133 95 57 31 3 1 0.5

231 TABLE 4 MASS S P E C T R A OF T H R E E Me3Si D I S A C C H A R I D E S F R O M P A R T I A L H Y D R O L Y SIS O F O V O M b C O I D

m/e

A

B

C

S t r u c t u r e (See ref. 4)

173 204

28 260

13 955

46 492

See text Me 3 Si--O--CH=

217

180

311

231

C H - - C H - - C H - - O - - M e 3 Si + O--Me3Si 451--Me3Si--OH G1+ GI--O--Me3Si--H+ ( n o n - r e d u c i n g t e r m i n a l ) Me3Si--O--CH=O--G1 + (reducing terminal) 668--Me3Si--OH See t e x t

361 451 539 569 578 583 668 683

100 38 5 6 -1

100 93 2 62 -20

100 73 6 22 1 21

---

---

---

CH--O--Me3Si

+

J

M+--(Me 3 Si--O--CH2--CH--O )--(Me 3 Si--O--CHMe 3Si--O--CH= C--CH= O--G1 +

O)

I O--Me3Si

m a n y as six peaks migrating in the region expected for Me3Si disaccharides (Table 1). A peak migrating at R~ = 1.83 (not shown in Table 1) has been shown by mass spectrometry n o t to be a Me3Si sugar. Approximately 1 t~g of peak B was collected and hydrolyzed as described in the Experimental section. GC analysis of the Me3Si monomers yielded only mannose, implying that disaccharide B is a mannobiose. The mass spectra of all the GC peaks in Tables 1--3 show strong peaks at m / e = 73 corresponding to (CH3)3Si ÷, a c o m m o n fragment in Me3Si compounds. The EI-MS of disaccharides A, B and C (Table 4) show strong peaks at m / e = 204 and 217 which are characteristic of the Me3Si derivatives of neutral sugars [4]. The peak at role = 173 which is relatively weak in the spectra of A, B and C is quite strong in the spectrum of pea~: D (not shown). This fragment, Me3Si--O--CH=CH--NHAc + is the strongest peak in the fragmentation of N-acetyl amino sugars [4,8]. Other peaks in the fragmentation of D at rn/e = 420 and 538 which m a y be assigned to sugar fragments containing the N-acetyl amino group, along with peaks at role = 204, 217 and 361 imply that peak D contains disaccharides composed of one neutral and one amido hexose [3,4,9]. Peak B2 is a mixture of disaccharides containing acetamido sugars which is n o t resolved on this column (SE-30) but which is partially resolved on OV-17. Partial methanolysis of ovomucoid results in a slightly more selective cleavage than does acid hydrolysis. In Table 2 we show that only two peaks appear but in slightly higher yield. The mass spectra of these two peaks show t h e m both to be disaccharides and the spectrum of t h a t at R~ = 1.12 shows it to be composed only of neutral hexoses (Table 5). Partial methanolysis of porcine gastric mucin gives three GC peaks which have been identified as Me3Si disaccharide methylglycosides. Mass spectra of two of t h e s e peaks

PGM a

130 52 0 30 71 47 12 0 36 0 16 41 4

420 451 480 490 511 521 539 552 553 583 625 637 782 814

117 29 40 31 27 24 0 0 18 0 0 14 8 7

70 0

866 89 1000 367 155

PGM R a = 1.76 65 ° C , 1 h

a PGM, p o r c i n e gastric m u c i n .

--

43 0

891 83 1000 448 229

65 ° C , l h

R a = 1.53

361 393

173 191 204 217 330

m/e

0 18 0 0 80 0 0 0 0 7 0 0 0 0

126 126

105 168 1000 526 0

Ovomucoid R a = 1.13 65 ° C , 2 h

Me3Si-O--~H= CH--NHAc+ Me3Si--O--CH--OMe3Si+ Me 3Si--O--CH= CH--OMe3Si + M e 3 S i - - O - - C H = CH--CH--OSiMe~ m/e 420--Me3SiOH (acetamido hexose) m/e 451--Me3SiOH (neutral hexose) GI--O--Me + ( n e u t r a l h e x o s e m e t h y l g l y c o s i d e ) G1NAc--O--SiMe~ ( a m i d o h e x o s e ) Gl--O--SiMe~ (neutral hexose) Me3Si--O--CH--O--G1NAac--OMe + (reducing end) M+(829)--(Me3Si--O--CH2 )--(Me3 S i - - O - - C H = CH--OSiMe 3)--Me OH Me3Si--O--CH--O--GI--OMe + (reducing end) M÷(829)--(Me3 Si--O--CH2 )--(Me 3Si--O--CH= C H - - N H A c ) - - M e O H n e u t r a l h e x o s e f r a g m e n t at r e d u c i n g e n d [ 3 ] 1 -+ 6 l i n k e d a m i d o h e x o s e [ 9 ] M+(829)--(Me3Si--O--CH2)--(Me3Si--O--CH=CH--NHAc) 1 -> 6 linked neutral hexose (nan-reducing end) M+(829)--Me3Si--O--CH=CH--OSiMe3 M+(829)--(Me~Si--O--CH2--CHO )--(MeO--CHO) M+(829)--(Me)--(MeOH) [ 9 ] M+(829)--(Me)

Structure assignment

MASS S P E C T R A O F Me3Si D E R I V A T I V E S O F M E T H A N O L Y Z A T E S

TABLE 5

872

same same same same same same 451 same same 538 same 569 same same same 611 same 683 same

Corresponding f r a g m e n t in Me3Si disaccharide

b~

b~

233

were adequate to identify them as being composed of one hexose and one acetamido hexose, a type of disaccharide which would be expected on the basis of the known carbohydrate structure of porcine gastric mucin [10]. DISCUSSION

The mass spectra of three disaccharides A, B and C isolated by partial hydrolysis of ovomucoid are sufficiently detailed to yield some s~ructural information. In the mass spectrum of mannobiose B (Table 4), the presence of a strong peak at r o l e = 583 corresponds to t h e fragment CH2 O SiMe3 J

/

MeaSiO ",,] SiMe3

\

// I OSiMe3

which is found only in the fragmentation of Me3Si derivatives of 1-~ 6 linked aldohexosyl aldohexoses [4]. We may conclude that disaccharide B is mannosyl (1 -~ 6) mannose. Although GC retention times are quite sensitive to anomeric configuration, the mass spectra are not. Therefore we c a n n o t determine the anomeric configuration of the 1 -> 6 linkage w i t h o u t additional information such as the relative retention times of a and ~ 1 -~ 6 linked mannobioses. The EI-MS of disaccharide C is the same within experimental error as that of B and we propose that it is simply the opposite anomeric Me3Si derivative of mannosyl (1 -> 6) mannose. The relative retention times of p e a k C and peak B are similar to those reported by Bhatti et al. [2] for the two anomeric Me3Si derivatives of Man(1 -~ 6)Man. Disaccharide A, like B and C has no detectable peak at r o l e = 668 which would be expected of disaccharides containing 1 -~ 2 or 1 -* 4 linkages [4]. Since the spectrum of A has only a small peak at r o l e = 583, we conclude that it is 1 -~ 3 linked. We propose t h a t this neutral disaccharide is mannosyl a(1 -~ 3) mannose in agreement with the results of Montreuil and collaborators [1] who have f o u n d by classical methods that mannosyl a(1 -~ 3) mannose and mannosyl a(1 -~ 6) mannose are the only neutral disaccharides in partial hydrolyzates of ovomucoid. The mass spectra of the Me3Si derivatives of the disaccharide m e t h y l glycosides in Table 5 can be interpreted by comparison to t h e mass spectra o f the Me3Si derivatives of monosaccharide methylglycosides and those of Me3Si disaccharides. A strong peak in spectra of the Me3Si derivatives of 2-acetamido sugars n o t substituted at C-3 occurs at r o l e 1 7 3 (Me3Si--O--CH=CH--NHAc+). The large peak at t h a t mass i n the two corn-

234

pounds from porcine gastric mucin along with peaks at m/e 420 and 330 imply that both peaks represent disaccharides containing acetamido sugars. The peaks at m/e 814 and 782 for these two disaccharides may be assigned as M~-I5 and M~-32-15 from a parent molecular ion at 829 corresponding to the Me3Si derivative of a disaccharide methylglycoside containing one acetamido hexose and one hexose. ~ons due to loss of methyl and methanol plus methyl are prominent in the spectra of methylglycosides of acetarnido sugars [9]. Assignment of most of the other fragment ions in the spectrum of these two compounds can be made by comparison to the spectra of Me3Si derivatives of amido sugar containing disaccharides. Ions retaining the reducing terminal methoxyl group occur 58 mass units below the corresponding fragmen~ in the spectra of Kammerling et al. [3], Thus the peak at m/e 625 (M+-204) resulting from the loss of the neutral fragment, Me3Si--O--CH=CH-O--SiMe3, is analogous to that found at m/e 683 by Kammerling etal. [3]. Since the peak at m/e 637 results from the loss of the reducing terminal carbon it occurs at the same mass in both our spectra and those of Kammerling e~ al. [3)° The absence of peaks at m/e 552 and 583 implies that neither of these disaccharides has a 1 -> 6 linkage. Unlike the porcine gastric mucin disaccharides~ the spectrum of that from o v o m u c o i d l a c k s f r a g m e n t s c h a r a c t e r i s t i c o f a c e t a m i d o sugars at m/e 1 7 3 , 4 2 0 a n d 3 3 0 b u t i t has f r a g m e n t s c o m m o n l y f o u n d in n e u t r a l h e x o s e s such as m/e 2 0 4 , 2 1 7 , 3 6 1 a n d 4 5 1 . F r a g m e n t s c h a r a c t e r i s t i c o f b o t h t h e r e d u c i n g t e r m i n a l (role 5 5 I , 3931 a n d t h e n o n - r e d u c i n g t e r m i n a l (role 5 8 3 ) r e s i d u e s c o r r e s p o n d m n e u t r a l h e x o s e s s u g g e s t i n g t h a t this GC p e a k c o r r e s p o n d s t o a d i s a c c h a r i d e m e t h y t g l y c o s i d e c o m p o s e d o f t w o n e u t r a l hexoses° T h e s t r o n g p e a k at role 583 is c h a r a c t e r i s t i c o f d i s a c c h a r i d e s h a v i n g a 1 - ~ 6 l i n k a g e [ 4 ] . I t seems l i k e l y t h a t t h i s GC p e a k r e p r e s e n t s m a n n o s e (1 -~ 6) m a n n o s e .

SIMPLIFIED DESCRIPTION

OF THE METHOD

AND

ITS APPLICATIONS

Although the approach described in this paper is unlikely to be as informative as methylation analysis, it can be done on a smaller sample and without conversion of the glycoprotein to glycopeptides. Further, the procedure is much easier to carry out, requiring less time than a methylation analysis. It is also possible that in certain cases this procedure could be used to resolve sequence ambiguities which might arise in methylation analysis. Comparison of partial acid hydrolysis and methanolysis shows the latter to be slightly more selective, giving fewer disaccharides in higher yields.

ACKNOWLEDGEMENT This research was supported by NIH

grants AI-II0i4

and GM-26336.

REFERENCES 1 Bayard, Bo, Strecker, G. and Montreuil, J. (1975) Biochimie 57, 155 2 Bhatti, T., Chambers, R.E. and Clamp, J.R. (1970) Biochim. Biophys. Acta 222, 339

235 3 Kammerling, J.P., Vliegenthart, J.F.G., Vink, J. and de Ridder, J. (1971) Tetrahedron 27, 4729 4 Kammerling, J.P., Vliegenthart, J.F.G., Vink, J, and de Ridder, J. (1971) Tetrahedron 27, 4275 5 Brobst, K.M. and Lott, C.E. (1966) Cereal Chem. 43, 35 6 Coduti, P.L. (1976) J. Chromat. Sci. 14,423 7 Montreuil, J., Adam-Chosson, A. and Spik, G. (1965) Bull. Soc. Chim. Biol. 47, 1867 8 Clamp, J,R., Dawson, G. and Hough, L. (1967) Biochim. Biophys. Acta 148,342 9 Coduti, P.L. and Bush, C.A. (1977) Anal: Biochem. 78, 21 10 Kochetkov, N.K., Derevitskaya, V.A. and Arbatsky, W.P. (1976) Eur. J. Biochem. 67,129