[39]

HIGH-MASS GC/IVIS OF PERMETHYLATED OLIGOSACCHARIDES

733

spectrometric techniques with other spectroscopic, enzymatic, chemical, and/or immunological methods represents the most direct and efficient way to establish frequent, yet unknown, molecular structure-function relationships. Acknowledgments We thank Dr. A. Vogel and Prof. K. Sandhoff, Bonn, for the sample of lysosulfatide. This work was generously supported by the Deutsche Forschungsgemeinschaft.

[39] H i g h - M a s s Gas C h r o m a t o g r a p h y - M a s s S p e c t r o m e t r y of P e r m e t h y l a t e d Oligosaccharides By

G U N N A R C . HANSSON a n d HASSE KARLSSON

Capillary gas chromatography-mass spectrometry (GC/MS) is a fast, sensitive, and high-resolving method for the characterization of biomolecules. It has been used in glycoconjugate research for analyzing the partially methylated alditol acetates after degradation of the glycoconjugates1 and, in a few cases, for the analysis of oligosaccharides after permethylation2-4 or trifluoroacetolysis. 5 The advent of high-temperature capillary GC using thin-film thermostable bonded stationary phases now allows the analysis of large permethylated oligosaccharides,6-8 an adaptation that should also be useful in other fields of biomedical research. Preparation of Oligosaccharides Glycosphingolipids

Glycan is cleaved7 from the ceramide portion of glycosphingolipids by endoglycoceramidase from Rhodococcus 9 (Genzyme Corp., Boston, MA) I This series, Vol. 50 [1]. 2 p. Hallgren and A. Lundblad, J. Biol. Chem. 252, 1014 (1977). 3 H. van Halbeek, L. Dorland, J. Haverkamp, G. A. Veldink, J. F. G. Vliegenthardt, B. Fournet, G. Ricart, J. Montreuil, W. D. Gathmann, and D. Aminoff, Eur. J. Biochem. 118, 487 (1981). 4 T. Tsuji and T. Osawa, Carbohydr. Res. 151, 391 (1986). 5 B. Nilsson and D. Zopf, Arch. Biochem. Biophys. 222, 628 (1983). 6 H. Karlsson, I. Carlstedt, and G. C. Hansson, FEBS Lett. 226, 23 (1987). 7 G. C. Hansson, Y.-T. Li, and H. Karlsson, Biochemistry 28, 6672 (1989). s H. Karlsson, I. Carlstedt, and G. C. Hansson, Anal. Biochem. 182, 438 (1989). 9 M. Ito and T. Yamagata, J. Biol. Chem. 261, 14278 (1986).

METHODS IN ENZYMOLOGY,VOL. 193

Copyright© 1990by AcademicPress. Inc. All fightsof reproductionin any formreserved.

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GLYCOCONJUGATES

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or ceramide-glycanase from the leach Macrobdella decora lo (Boehringer Mannheim, Mannheim, FRG). The released oligosaccharides are obtained in the water phase after partitioning with chloroform:methanol: water, 8 : 4 : 3 (by vol) and residual detergents trapped by Sep-Pak 7 (Waters, Milford, MA). The ceramides and monoglycosylceramides are obtained in the chloroform phase. 7 O-Linked Oligosaccharides The mucins or other glycopeptides (I mg/ml) are treated with 0.05 M KOH and 1.0 M NaBH4 at 45 ° for 16 or 45 hr. 6,8,11 Acetic acid (2 M) is added, the sample passed over an ion-exchange column (AG 50W-X8, H ÷, Bio-Rad, Richmond, CA; 0.5 g resin/ml sample) eluted with water (10 ml/g resin), and evaporated four times after the addition of methanol with a few drops of acetic acid. The neutral oligosaccharides are separated from the acidic ones by ion-exchange chromatography on DEAE-Sephadex. 6,a Permethylation The oligosaccharides are dried in a stream of nitrogen in tubes with a Teflon-faced screw cap, dried under vacuum for 30 min, and permethylated s,12,13 by the addition of 0.5 or 1.0 ml (less than 0.2 mg and 0.3-2 mg of sample, respectively) of dimethyl sulfoxide (DMSO), 0.1 or 0.2 ml of iodomethane (0.5 or 1.0 ml for oligosaccharides with a Glc-alditolS), and 25 or 50 mg of NaOH powder. The samples are stirred with a magnetic bar for 10 min and the reaction stopped by the addition of 2 or 4 ml of water and 1 or 2 ml of chloroform. The water phase is removed after centrifugation and the chloroform phase washed ->4 times (until neutral) with 2 or 4 ml of water. The chloroform phase is moved to a new tube and evaporated. Oligosaccharides with a reducing end, as obtained from glycosphlngolipids, can be reduced with 0.5 ml 1 M NaBH4 overnight, evaporated with nitrogen, and the last step repeated 5 times after the addition of methanol and a drop of acetic acid. The reduced oligosaccharides are dried under vacuum and permethylated as above, but with increased amounts of iodomethane (0.5 or 1.0 ml). s

10 S.-C. Li, R. DeGasperi, J. E. Muldrey, and Y.-T. Li, Biochem. Biophys. Res. Commun. 141, 346 (1986). 11 D. M. Carlsson, J. Biol. Chem. 243, 616 (1968). 1~I. Ciucanu and F. Kerek, Carbohydr. Res. 131, 209 (1984). 13 G. Larson, H. Karlsson, G. C. Hansson, and W. Pimlott, Carbohydr. Res. 161, 281 (1987).

[39]

HIGH-MASSGC/MS OF PERMETHYLATED OLIGOSACCHARIDES

735

Preparation of Capillary Columns Capillary columns are prepared from uncoated and deactivated, fusedsilica capillary tubing (0.25 mm id) with an outside coating of HT-polyimide, intended for use up to 430° (Chrompack, Middelburg, The Netherlands). Columns (5 or 10 m) are statically coated (0.03-0.05 t~m) with freshly made stationary-phase solutions of SE-54 (Alltech, IL) in dichloromethane/pentane, 1 : 1 (by vol) containing 0.8% (w/w stationary phase) dicumyl peroxide (Merck, Darmstadt, FRG). After coating, the capillaries are flushed with dry nitrogen for 30 rain at room temperature and then evacuated by attaching both ends to vacuum. The columns are then sealed with a microflame and cross-linked TMby heating to 145° for 30 rain. Columns are then rinsed with 5 ml of dichloromethane and conditioned at 380° for 4 hr using hydrogen as carrier gas. C o n d i t i o n s for G a s C h r o m a t o g r a p h y - M a s s

Spectrometry

A Carlo Erba 4160 gas chromatograph was connected to a magnetic sector instrument (VG ZAB-HF, data system VG 11-250) with a homemade interface allowing temperatures up to 400 °. The fused-silica columns (10 or 5 m) were introduced directly into the ion source and the tip positioned 1-2 mm from the electron beam. Helium was used as carrier gas with a head pressure of 0.15 bar and a linear gas velocity of 90 cm/sec at 87° for a 10-m column. A high-capacity gas purifier and an OMI-1 indicating purifier (both Supelco, Bellefonte, PA) were used in the carrier gas line. Samples dissolved in ethyl acetate were injected (1/~l; 1-100 ng/component) by the on-column technique. Injections were done at 87° or 200° and after a 1-min isothermal period the temperature was increased 10°/rain up to 3700-400 ° and held for 5 rain. The following conditions were used for the mass spectrometer: interface and ion source, 365°-400°; electron energy, 70 eV; trap current 500/~A; accelerating voltage, 8 kV; mass range scanned, m/z 1600-160; scan speed, 2 sec/decade; total cycle time, 3.5 sec; resolution 1400; pressure in the ion source region, l0 -5 mbar. Fomblin was used as calibrating substance using the direct inlet system. All spectra were background subtracted. Examples and Comments

Short capillary columns for GC have allowed the analysis of large permethylated oligosaccharides with up to 11 sugar residues and molecular masses up to 2300. 6-8 Due to the special coupling problems (cold and hot 14L. Blomberg, J. Buijten, K. Markides, and T. W~innman, J. Chromatogr. 239, 51 (1982).

736

GLYCOCONJUGATES

[39]

2_19-32 187 189 I@@

J

228 M=1642.8 2191 5971 8421 1219 I I I I Hex~O-Hex+O+HexNAc+O-Hex-O+Hex I I I 0 1103O 0

376-32 344

189

5'76 .,..;

-x3

i I

1030

1030-376+1 597-32 654 IILt~ ~J,~,l.J ....... la.l . . . . . . ~m

m

1. ~

m/z

1584 tM

'

ten

Wm

~

Fro. 1. Mass spectrum from GC/MS of a permethylated oligosaccharide released by ceramide-glycanase from a blood group B-active ganglioside based on GMI obtained from rat stomach, m5A 10-m column coated with 0.05/zm stationary phase was used, injection at 200°, temperature raised 10°/min up to 390° where the mass spectrum reproduced was recorded.

spots, length of interface, problems with short columns, etc.), it has not yet been possible to reach the equivalent size using GC/MS (the limit at present is eight to nine sugars). A longer column with a length of 10 m can be used for analyses up to seven to eight sugar residues and gives good resolution. A shorter column (5 m) can be used for larger saccharides using faster temperature programs to enhance the resolution and minimize the elution time. Evidence for thermal degradation of the permethylated oligosaccharides has been observed with longer elution times and temperatures higher than 400 °. Oligosaccharides released from neutral glycosphingolipids and gangliosides have been successfully analyzed, 7 as well as similar oligosaccharides found in free form in, for example, milk. Figure 1 shows a mass spectrum

[39]

100¸

HIGH-MASS G C / M S OF PERMETHYLATED OLIGOSACCHARIDES M=5ll H-HNol

737

M=1176 HN-H-HN-HNol I F

M=685 F-H-HNol M=552 HN-HNol

I

M=756 H HNHNol " "

M=931 F-H-HNoHNol M=931 I HN-H-HNol I

L

IB Jl

m/z 228

M=552

/

J

H-HNoI-HNII IHN-HNoI-HN|I /

/

M1002

/

l

"

IL,I_)IL

..

TIC

H-HNol-HN-H

,

3~II j FI'H-HN°/I-HN'H'F

"

/

/

J_ /

lOO¸

1276

260

/

M=I175.6

I CH20Me I 260I 638I 883l HC-~-CO-CHJ I I 11 J HexNAc.FO-Hex+O.FHexNAc.FO-I-CH i I I 1521 HC-OMe ..-J . . . . . . 1087 189 Fu 0 HC-OMe I

CH2OMe 883

~x5

-.~

-x20

2~8 ~6 }9 96 ! 723 883-32 851

521 576 6O6 418

LI 2oo

,.L

,

4~

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I

. . . . . . . . .

m/z

M-145 1031 M-89 1087 M-45 .

o

FIc. 2. GC/MS of permethylated neutral oligosaccharides released from porcine small intestinal mucin glycopeptides. The partial total ion current is shown on top with the major oligosacchafide structures marked. A portion of the chromatogram is enlarged (on the fight-hand side) which shows a mass chromatogram of m/z 228 (260-32) and part of a gas chromatogram of the same sample. H, Hexose; HN, N-acetylhexosamine; F, fucose; HNol, for N-acetylhexosaminltol (from the GalNAc linked to the peptide). The sequence to the left of HNol is finked to C-3 and that to the fight to the C-6 of the HNol. The mass spectrum of scan 311, the middle peak in the cluster, is shown at the bottom of the figure.

738

GLYCOCONJUGATES

[40]

from GC/MS of a seven-sugar oligosaccharide containing sialic acid obtained from a purified blood group B ganglioside. 15This eluted at 390° on a 10-m column. The endoglycoceramidase or ceramide-glycanase enzymes do not split monoglycosylceramides, but these small intact glycosphingolipids can be analyzed by GC/MS. 7 Oligosaccharides released from mucins and other glycoproteins by alkaline-NaBH4 treatment can be analyzed and the high resolution achieved is necessary for separating the complex mixtures found in nature. 6"s An example from neutral oligosaccharides of porcine small intestinal mucins is shown in Fig. 2, where three isomeric pentasaccharides are resolved and characterized. Permethylation using solid NaOH gives good yields for neutral oligosaccharides with a GalNAc-alditol, and neutral and sialic acid-containing oligosaccharides with a Glc-aldose. Oligosaccharides with a Glc-alditol give lower yields by permethylation, although these can be increased by modifying the permethylation procedure s (see above). Sialic acid-containing oligosaccharides with a GalNAc-alditol give poor yields. Acknowledgments This work was supported by grants from the Swedish Medical Research Council (No. 7461), the SwedishBoard for Technical Development,and Gabrielsson's research fund. The mass spectrometer was supported by the Swedish Medical Research Council (No. 3967). 15J.-F. Bouhours, D. Bouhours, and G. C. Hansson, J. Biol. Chem. 262, 163700987).

[40] T a n d e m

Mass Spectrometry of Glycolipids

By CATHERINE E. COSTELLO and JAMES E. VATH

Introduction The structural determination of glycolipids has been considerably advanced through the implementation of mass spectral methods, because of their sensitivity and the wealth of information they convey. There are, however, practical limitations to the utilization of the usual mass spectrometry-based approaches for the analysis of very small quantities of material, particularly when the sample consists of a mixture of closely related compounds. Tandem mass spectrometry (MS/MS) offers a means to adMETHODS IN ENZYMOLOGY, VOL. 193

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