[36]

MS OF CHROMOPHORE-COUPLED OLIGOSACCHARIDES

661

[36] D e s o r p t i o n M a s s S p e c t r o m e t r y of Oligosaccharides Coupled with Hydrophobic Chromophores

By LINDA POULTER and A. L. BURLINGAME Introduction Early strategies involving mass spectrometric investigation of carbohydrates required chemical derivatization such as permethylation of all labile hydrogens to permit sample vaporization without thermal decomposition for use of electron impact and chemical ionization methods and their GC/MS analogs. ~ Naturally occurring partial methylation and complete chemical methylation was also found to facilitate studies of high mass oligosaccharides using field desorption. 2'3 Karlsson and co-workers exploited a variety of derivatization strategies in order to permit vaporization and also to direct fragmentation in their pioneering systematic studies of high-mass glycolipids.4,5 These methods and issues have been reviewed elsewhere. 6,7 Since 1980, liquid matrix sputtering/ionization methods have revolutionized the utility of mass spectrometric-based strategies for the structural characterization of biopolymers. 8 Virtually all classes of labile, polar biological substances may now be investigated throughout an extended mass range (< 10,000 Da). This includes a wide variety of structural classes of oligosaccharides occurring as components of natural products, glycolipids, glycoproteins, and proteoglycans. Although early studies using this new desorption ionization method were carried out on free oligosaccharides9 or the corresponding oligoglyt T. Radford and D. C. de Jongh, in "Biochemical Applications of Mass Spectrometry (G. R. Waller and O. S. Dermer, eds.), 1st Suppl. Vol., p. 256. Wiley (Interscience), New York, 1980. -' M. Linsheid, J. D'Angona, A. L. Burlingame, A. Dell, and C. E. Ballou, Proc. Natl. Acad. Sci. U.S.A. 78, 1471 (1981). 3 A. L. Burlingame, C. E. Ballou, and A. Dell, Proc. 29th Annu. Conf. Mass Spectrom. Allied Topics, Minneapolis, MN, p. 528 (1981). 4 K.-A. Karlsson, Frogr. Chem. Fats Other Lipids 16, 207 (1977). 5 B. E. Samuelsson, W. Pimlott, and K.-A. Karlsson, this volume [34]. 6 V. N. Reinhold and S. A. Can', Mass Spectrom. Rev. 2, 153 (1983). 7 A. Dell and G. N. Taylor, Mass Spectrom. Rev. 3, 357 (1984). s A. L. Budingame and J. A. McCloskey (Eds.), "Biological Mass Spectrometry." Elsevier, Amsterdam, 1990. 9 j. p. Kamerling, W. Heerma, J. F. G. Vliegenthart, B. N. Green, I. A. S. Lewis, G. Strecker, and G. Spik, Biomed. Mass Spectrom. 10, 420 (1983).

METHODS IN ENZYMOLOGY, VOL. 193

Copyright ~ 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

662

GLYCOCONJUGATES

[36]

coalditols, 6,9-H it has gradually been recognized that such particularly hydrophilic substances present virtually a worst-case scenario for the optimal exploitation of energetic particle-induced sputtering/ionization from the surface layer of viscous hydrophilic liquid matrices such as glycerol, thioglycerol, or triethanolamine. This situation is due both to the lack of surface activity of free carbohydrates in these liquids and to the difficulties in complete separation of such compounds from biological matrices and salts. Hence early recipes advocated the careful addition of alkali metal salts when a sample did not work to form alkali metal attachment ions, ~2 which provided a charge and improved the surface activity compared with the corresponding free oligosaccharide. Thus, [M + alkali metal ion] ÷ species are observed in the positive ion mode and the corresponding [M + halide ion]- species in the negative ion mode. 13 These studies typically required 1-50 nmol of sample per component. 9,H Subsequently, in order to ease the problems of isolation and salt removal as well as increase the sensitivity and promote fragmentation at certain preferred sites, a considerable amount of attention has been directed toward the use of both permethylation and peracetylation combined with sputtering ion soruces, namely, cesium ion liquid secondary ion mass spectrometry (LSIMS) and xenon neutral fast atom bombardment ( F A B ) . 7'11'14-17 These derivatives usually permit the investigator to obtain sufficient mass spectral data to determine some features of the structural class and degree of homogeneity of the sample. However, when such derivatives give poor quality signals, sodium acetate or ammonium thiocyanate is often added to the liquid matrix. ~7While it is clear that permethyl and peracetyl derivatization eases the difficulties of isolation and salt removal, these derivatives are of little use in subsequent separation of components of mixtures. Also, little mention is usually made of the fact _that both derivatization procedures yield underderivatization. ~.~-~9This, of 10A. L. Burlingame, in "Secondary Ion Mass Spectrometry, SIMS IV" (A. Benninghoven, J. Okano, R. Shimizu, and H. W. Werner, eds.), p. 399. Springer-Vedag, Berlin, 1984. ii j. Peter-Katalinic and H. Egge, Mass Spectrom Rev. 6, 331 (1987). 12C. Bosso, J. Defaye, A. Heyraud, and J. Ulrich, Carbohydr. Res. 125, 309 (1984). 13 D. Prom~, J. C. Prom6, G. Puzo, and H. Aurelle, Carbohydr. Res. 140, 121 (1985). 14 S. A. Carr, G. D. Roberts, and M. E. Hemling, in "Mass Spectrometry of Biological Materials," (C. N. McEwen and B. S. Larsen, eds.), p. 87. Dekker, New York, 1990. 15 A. Dell, Adv. Carbohyd. Chem. Biochem. 45, 19 (1987). 16 A. Dell and J. E. Thomas-Oakes, in "Analysis of Carbohydrates by GLC and MS" (C. J. Biermann, and G. D. McGinnis, eds.), p. 217. CRC Press, Boca Raton, FL, 1988. 17 A. Dell, this volume [35]. 18 G. J. Gerwig, P. de Waard, J. P. Kammerling, J. F. G. Vliegenthart, E. Morgenstern, R. Lamed, and E. A. Bayer, J. Biol. Chem. 264, 1027 (1989).

[36]

MS OF CHROMOPHORE-COUPLEDOLIGOSACCHARIDES

663

course, distributes the available total ion current among several pseudomolecular ionic species of the particular component being analyzed j8 and, in effect, compromises the potential gain in overall sensitivity expected from the increased hydrophobicity conveyed by derivatization. In addition, such derivatives increase the molecular weight significantly which results in lowering sensitivity a s well. 15"16"2°-22 While use of permethylation and peracetylation provides a convenient strategy to obtain structural class information on carbohydrates attached to glycoproteins and glycolipids without the need for rigorous sample purification, 14,15,21,23relatively little attention has been devoted to development of chemically and instrumentally integrated strategies including isolation, separation, and derivatization, which would also provide inherently optimized mass spectral quality and sensitivity. While bringing about such an integrated approach is a formidable interdisciplinary challenge, there is still a great need for methodology which could provide rigorous structural characterization of individual carbohydrate isomers at the subnanomole level. This is the subject of the remainder of this chapter. In this laboratory we have focused attention on the systematic exploration of the use of reductive amination derivatization strategies to effect coupling a hydrophobic chromophore with the free reducing terminus of the chitobiose core upon liberation of asparagine-linked glycosylation by enzymatic treatment of glycoproteins with endoglycosidase H (Endo H) and peptide-N4-(N-acetyl-fl-glucosaminyl) asparagine amidase F (PNGase F). Our initial efforts were devoted to studying the mass spectral fragmentation behavior of the oligoglycoaminodeoxyalditols formed using the paminobenzoic acid ethyl ester (ABEE) in both positive and negative ion mode and to establish the characteristics of the spectrum-structure correlations. 24 We discovered that the negative ion mode of fragmentation elaborates an unusually abundant, structurally informative ion series. The relative abundances of the fragments observed were shown to correlate with oligosaccharide residue branching patterns. This important finding permitted us to identify the N-linked glycosylation on human hepatitis B ~ G. O. Aspinall, in "The Polysaccharides," (G. O. Aspinall, ed.), Vol. l, p. 49. Academic Press, New York, 1982. 20 A. Dell, Biomed. Environ. Mass Spectrom. 16, 19 (1988). 21 A. Dell, Biochimie 70, 1435 (1988). 22 H. Sasaki, N. Ochi, A. Dell, and M. Fukuda, Biochemistry 27, 8618 (1988). _'2 A. L. Burlingame, D. Maltby, D. H. Russell, and P. T. Holland, Anal. Chem. 60, 294R (1988). J. W. Webb, K. Jiang, B. L. Gillece-Castro, A. L. Tarentino, T. H. Hummer, J. C. Byrd, S. J. Fisher, and A. L. Burlingame, Anal. Biochem 169, 337 (1988).

664

GLYCOCONJUGATES

[36]

major glycoprotein surface antigen25 and the high mannose, hybrid, and complex components of glycosylation attached to subunits of nicotinic acetylcholine receptor. 26,27 In addition, the use of this strategy together with the benefit of the knowledge obtained from the systematic investigation of this class of derivative discussed herein has led recently to assignment of a new Saccharomyces cerevisiae mnn mutant N-linked oligosaccharide structure in which the ManaGlcNACE-core oligosaccharide is enlarged by addition of the outer chain to the t~l-->3-1inked mannose in the side chain that is attached to the fll--+4-1inked mannose rather than by its addition to the terminal al--~6-1inked mannose. 2a We have reported further results of a systematic study of increasing alkyl ester chain length and its concomitant enhancement of relative hydrophobicity yielding up to a factor of 40 increase in LSIMS sensitivity. 29,3° The importance of significantly increased sensitivity coupled with the previously noted advantages of a derivative class containing a chromophore permitting HPLC detection and effective isolation and desalting is further demonstrated in this work by presentation of results vide infra obtained from further investigation of the glycosylation of four related glycoproteins which comprise the nicotinic acetylcholine receptor from Torpedo californica. This was carded out employing the particular derivative optimized for coupling efficiency and LSIMS sensitivity using the wide diversity of structures expressed at low molar levels on a 250-/zg (1 nmol) sample of intact acetylcholine receptor. Methods

Synthesis of N-Alkyl p-Aminobenzoates N-Hexyl, n-octyl, and n-decyl p-aminobenzoates are prepared with slight variations according to Kadaba et al. 31 The appropriate alcohol (0.1-0.15 mol) is added to p-aminobenzoic acid (0.01 mol) in the presence 25 B. Gillece-Castro, S. J. Fisher, A. L. Tarentino, D. L. Peterson, and A. L. Burlingame, Arch. Biochem. Biophys. 256, 194 (1987). 26 L. PouRer, J. P. Earnest, R. M. Stroud, and A. L. Burlingame, Biomed. Environ. Mass Spectrom. 16, 25 (1988). 27 L. Poulter, J. P. Earnest, R. M. Stroud, and A. L. Burlingame, Proc. Natl. Acad. Sci. U.S.A. 86, 6645 (1989). 28 L. M. Hernandez, L. Ballou, E. Alvarado, B. L. Gillece-Castro, A. L. Burlingame, and C. E. Ballou, J. Biol. Chem. 264, 11849 (1989). 29 L. Poulter, R. Karrer, K. Jiang, B. L. Gillece-Castro, and A. L. Burlingarne, Proc. 36th Annu. Conf. Mass Spectrom. Allied Topics, San Francisco, CA, p. 921 (1988). 30 L. Poulter, R. Karrer, and A. L. Burlingame, Anal. Biochem., submitted. 31 p. K. Kadaba, M. Carr, M. Tribo, J. Triplett, and A. C. Glasser, J. Pharmacol. Chem. Sci 58, 1422 (1969).

[36]

MS OF CHROMOPHORE-COUPLEDOLIGOSACCHARIDES

665

of boron trifluoride ethyl etherate (0.015 mol, 48%). The resulting suspensions are refluxed for between 10 and 24 hr until all of the acid is consumed. The excess alcohol is removed by distillation under reduced pressure (10-100/zm Hg). The products are dissolved in 25-40 ml of ether, washed with sodium carbonate solution (80 ml, 5%), and washed with distilled water (2 x 80 ml). The organic phase is then treated with hexane (300-500 ml) and crystallized overnight at - 2 0 °. Recrystallization from hexane provides the desired esters (yield 61-85%). N-Tetradecyl p-aminobenzoate is prepared according to the description of Flynn et a1.,32 by reacting p-nitrobenzoyl chloride (0.05 mol) with n-tetradecanol (0.05 mol) and pyridine (1 ml) in ethanol (100 ml) under reflux for 8 hr. The solvent is removed under reduced pressure and the crude ester is dissolved in ether and washed with sodium carbonate solution (80 ml, 10%) until free from p-nitrobenzoic acid. The ethereal solution is then washed with water (2 x 80 ml) and the ether evaporated. The resulting ester is then reduced under a hydrogen atmosphere in the presence of Pearlman's catalyst (palladium hydroxide on carbon, 0.05 g) at 60o-70° over 24 h to give an overall yield of 42% after recrystallization from hexane.

Preparation of Oligosaccharides for Derivatization This method is intended to be incorporated as an integral part of our analytical strategy when only very limited quantities of glycoprotein are available for analysis. Oligosaccharides are released from N-linked glycosylation sites on the glycoprotein (1-2 mol) by digestion with peptideN-glycosidase F (PNGase F). 33 The amount of enzyme needed varies considerably with the nature of the glycoprotein substrate. For example, in our experience 1 mU (milliunit) enzyme/30/zg protein is capable of digesting ribonuclease B, whereas 1 mU enzyme/5/.~g protein is required for digestion of t~racid glycoprotein and the nicotinic acetylcholine receptor from Torpedo californica. 27'3°Digestion is carried out in ammonium bicarbonate buffer (100 mM, pH 8.6) with 0.1% sodium dodecyl sulfate (SDS) and 0.6% Nonidet P-40 (NP-40) at 37° for 18 hr. Analytical SDS-PAGE, according to Laemmli, 34 on an aliquot of the digestion mixture with Coomassie staining (or silver staining for most economic use of sample) is used to show that digestion is complete. SDS present in the digestion mixture is then precipitated by adding guanidine-HCl (I M) 32 G. L. Flynn and S. H. Yalkowski, J. Pharmacol. Chem. Sci. 61, 838 (1972). 33 A. L. Tarentino, C. M. Gomez, and T. H. Plummer, Jr., Biochemistry 24, 4665 (1985). U. K. Laemmli, Nature (London) 227, 680 (1970).

666

o

HOH2Cx,r__0

Oo oO.

[36]

GLYCOCONJUGATES

H2N

~--OR

CH20H J~OH

~

O

NaCNBH3; H+/H20/MeOH 80°, 30-45 Min

R = Me, Et, Bu, Hex, Oct, Dec, Tdec SCHEME 1. Oligosaccharide derivatization.

dropwise,35 and after refrigerating for 2 to 4 hr to ensure complete precipitation, the suspension is centrifuged and the supernatant carefully removed with a micropipette and applied to a C~8 reversed phase Sep-Pak cartridge (Millipore-Waters, Bedford, MA). Oligosaccharides and salt are eluted by washing with water, leaving the protein and residual detergent (NP-40) absorbed on the cartridge packing. The aqueous sample is then lyophilized for subsequent derivatization. In our experience with subunits of the nicotinic acetylcholine receptor, glycoproteins which have been purified by preparative SDS-PAGE and isolated by electroelution are amenable to the above approach with subsequent derivatization of oligosaccharides for mass spectrometry; but to ensure success; the electroeluted protein should first be subjected to ethanol or acetone precipitation 36or extensive microdialysis to remove aqueous contaminants also concentrated by the electroelution process. Care too should be taken not to overload the C18 Sep-Pak cartridge, otherwise the removal of detergent and protein will not be complete. Cartridges can be used in series if more than microgram quantities of glycoprotein are digested, and in all cases the protein may be eluted separately with, for example, 80% acetonitrite/0.1% TFA (v/w) for subsequent analysis.

Oligosaccharide Derivatization Oligosaccharide (1-20/zg) is dissolved in water (40/~1) in a glass Reactivial (Pierce Chemicals, Rockford, IL) (5-ml capacity) that has previously been treated with silylation reagent (Pierce Chemical). n-Alkyl p-aminobenzoate (0.I mmol), sodium cyanoborohydride (35 mg), glacial acetic acid (41/zi), and methanol (350/~1) are mixed separately in a glass vial to form the reagent mixture (see Scheme 1). Warming of the vial will aid 35 j. E. Shively, in "Methods of Protein Microcharacterization" (J. E. Shively, ed.), p. 41. Humana Press, Clifton, NJ, 1986. 36 W. H. Konigsberg and L. Henderson, this series, Vol. 91, p. 254.

[36]

MS OF CHROMOPHORE-COUPLED OLIGOSACCHARIDES

667

solubility of n-hexyl and n-octyl p-aminobenzoates, while the substitution of methanol (120/~1) by an equal volume of chloroform is recommended to solubilize n-decyl and n-tetradecyl p-aminobenzoates. Reagent mixture (40/~!) is added to the solution of oligosaccharide in water and the total volume of the reaction mixture is then made up to 200 tzl with methanol. A chloroform/methanol (1 : 2, v/v) should be used in place of methanol when n-decyl and n-tetradecyl p-aminobenzoates are used. The Reactivial is sealed with a Teflon-lined cap, vortexed, and heated at 80°. After 45 min the vial is cooled and distilled water (1 ml) is added. Chloroform (1 ml) is then added when ethyl, n-butyl, n-hexyl or n-octyi p-aminobenzoates are used as derivatizing agents. Hexane (1 ml) is recommended when n-decyl and n-tetradecyl esters are used for derivatization. The mixture is vortexed and then centrifuged to efficiently separate the two phases. The aqueous phase containing the derivatized oligosaccharide is removed and the organic phase is reextracted with water (1 ml). Aqueous phases are combined and lyophilized. The absolute yield of the ethyl ester reductive amination with maltoheptaose (M7) was measured by GC/MS of the trimethylsilyl methylglucose formed from methanolysis and found to be approximately 60%. Recent studies in this laboratory have lead to a modified protocol for carrying out this reaction using a chitobiose reducing terminal oligosaccharide with a variety of hydrophobic chromophores in similar high yield. 37

Reversed-Phase Chromatography of Derivatized Oligosaccharides Derivatized oligosaccharides are both separated and effectively desalted by reversed-phase chromatography using a C~8 or C4 column (Vydac, 25 cm x 4.6 mm or 25 cm x 2.1 mm are used by the authors) and a water/acetonitrile solvent system with a gradient of 0 to 60% acetonitrile in 60 min and a flow rate of 1 ml/min or 300/.d/min, depending on the column i.d. A C~a column is recommended for highly acidic oligosaccharides, while a C4 column is best suited for neutral oligosaccharides derivatized with the more hydrophobic n-alkyl p-aminobenzoates. The oligosaccharides are monitored by their absorbance at 304 nm, which is conferred by the p-aminobenzoate group. Peaks may be integrated to provide an estimate of the relative amounts of oligosaccharide present since only one chromophore is present per molecule of oligosaccharide. Eluted samples are either lyophilized or reduced in volume by vacuum centrifugation prior to mass spectrometric analysis. 37 S. Kaur, W. Liang, R. R. Townsend, and A. L. Burlingame, Anal. Biochem., in preparation.

668

GLYCOCONJUGATES

[3 6]

Liquid Secondary Ion Mass Spectrometry (LSIMS) of Derivatized Oligosaccharides Samples ofderivatized oligosaccharides, typically submicrogram quantities, are applied to a matrix of glycerol/thioglycerol, 2 : 1 (v/v) (1/.d) on the probe tip and excess solvent pumped away after initial evacuation in the mass spectrometer. Trifluoroacetic acid (1%) may be added to the matrix to aid protonation when positive ion spectra are required. Positiveion LSIMS yields predominantly molecular weight data only, while negative ion spectra show extensive fragmentation. Higher sensitivities can be obtained by using a smaller volume of matrix, 3s but the duration of the ion signal is shortened significantly, thereby making it unsuitable for the recording of a complete spectrum of a large oligosaccharide unless multichannel array detection is available on MS-1. However, if the approximate size of the oligosaccharide is known and only molecular weight data is required, positive ion spectra may be recorded over the molecular ion region successfully using this approach with a postacceleration detector only. Spectra are recorded with the magnet in field control mode, scanning at either 100 or 300 sec/decade and at a mass resolution of 2000-3000. Mass calibration is performed using a mixture of Ultramark 1621 and 443 (PCR Research Chemicals Inc., Gainesville, FL). Results

Maltoheptaose In studies aimed at optimizing sensitivity in the analysis of oligosaccharides by LSIMS, we have investigated the relative sputtering efficiencies of a series of alkyl esters ofp-aminobenzoic acid by reductive amination of maltoheptaose. Those chosen include ethyl p-aminobenzoate, the derivatizing agent used in many of our earlier studies. Six additional esters were synthesized, including the methyl, n-butyl, n-hexyl, n-octyl, n-decyl, and n-tetradecyl, and coupled with the heptasaccharide maltoheptaose. Figures la and lb show the variation in the protonated molecular ion abundances of the various derivatives of maltoheptaose as a function of the time spent by the sample in the primary cesium ion beam, using 1.0 and 0. I /zg of derivative, respectively, dissolved in 1 /~1 of matrix (thioglycerol : glycerol, 1 : 1). It can be seen that there is a steady increase in the molecular ion abundances with increasing alkyl chain length, the effect being most pronounced as the concentration of derivatized oligosaccharide in the liquid matrix is decreased. For example, after 50 sec in the 3s H. Kambara, in "Mass Spectrometry in the Health and Life Sciences" (A. L. Burlingame and N. Castagnoli, Jr., eds.), p. 65. Elsevier, Amsterdam, 1985.

[36]

669

M S OF CHROMOPHORE-COUPLED OLIGOSACCHARIDES

60

(a)

5o •~ 4. 4,. 4•o. .e-

40 o vJ

M7-ABME M7-ABEE M7-ABBE M7-ABHE M7-ABOE M7-ABDE M7-ABTDE

o~

o

Z3o

,P,,i

r,¢l 2 0

10 ¸

0

i

0

I O0

i

200

i

2500

i

400

i

500

600

T i m e / sec FIG. 1. Protonated molecular ion abundances of n-alkyl p-aminobenzoates with respect to the chemical noise as a function o f time spent in the primary ion beam for (a) 1.0/~g and (b) 0.1/zg o f derivatized M7 in 1.0 v.l o f thioglycerol: glycerol (1 : 1). Plotted points represent the mean of three independent sets o f readings taken at fixed time intervals.

primary beam, it can be seen that 0.1/zg of M7 methyl p-aminobenzoate is not discernible above the level of the chemical noise, whereas M7 ntetradecyl p-aminobenzoate provides a maximum signal-to-noise ratio of 40 : 1. Relative molecular ion abundances were similar in the negative ion mode (data not shown), 0.1/~g of the methyl ester showing once again a signal barely above the level of the noise, while the tetradecyl ester gave an excellent molecular ion as well as distinct fragment ions. Dilution experiments showed that molecular ions for n-decyl p-aminobenzoate and n-tetradecyl p-aminobenzoate could be obtained at concentrations of 0.01 /~g//.d matrix, the latter having a maximum signal-to-noise of 7 : 1, while even at a concentration as low as 0.001/.,g//zl a weak molecular ion for the

670

GLYCOCONJUGATES

[36]

50

(b)

40

/•

¸

cn .m4 0 30

-e4..o4-

M7-ABEE M7-ABBE M7-ABHE M7-ABOE M7-ABDE - BTDE

Z

t~ r~ 2 0 '

I0'

.

•

.

.

.

•

,

.

.

i

0

w

I00

200

300

T i m e / see FIG. 1. (continued)

tetradecyl ester was observed. Although the sensitivity achieved with n-tetradecyl p-aminobenzoate is impressive, unfortunately, the coupling yields are low, 3° especially with more hydrophilic oligosaccharides (e.g., large N-linked structures and acidic species), which are not particularly soluble in methanol. In practice, it has been found that n-octylp-aminobenzoate provides the optimum alkyl chain length to enhance mass spectral sensitivity while maximizing the yield in preparation of the derivative. The negative ion mass spectrum of 2.0/zg of M7 n-octyl p-aminobenzoate is shown in Fig. 2 to illustrate mass spectral fragmentation characteristics. The fragment ion nomenclature used is according to Domon and Costello 39 and is detailed in Schemes 2a and 2b. It should be noted that a relatively abundant smooth profile of ions (Y) are observed from sequential elimination of anhydro moieties from the nonreducing terminus. In addition, a series of nonreducing terminal charge-bearing ions (2.4A-type) are present. 39 B. D o m o n and C. E. Costello,

Glycoconjugate J.

5, 397 (1988).

[36]

MS OF CHROMOPHORE-COUPLED OLIGOSACCHARIDES

671

(M-H) 1384

Y 412

Z,4A

y

/I 3i3 [ i

i

'"

: :~

736 z,7i[1' A 1,

Z'5A4557i41 '

''

'

,

y

':

;

i !

~

Y

~'4A8691898/ !

;

:

' i :1

1060 Y 1112721222 i

*, ,!,.,.I.,~.l',

:'i;dl : r':

il

.:1:. :l:l:l:J!giill;t~lliIZ!lliitjllilltll~lUIlflllllmih

FIG. 2. Negativeion LSIMS mass spectrumof 2.0/zg of n-octylp-aminobenzoatein 1.0 p.lofthioglycerol: glycerol(1: 1). Fragmentionsoftype Yarisefromglycosidicbondcleavage withhydrogentransferand chargeretentionat the derivatizedreducingterminus(seeScheme 2a), whilethose designated2'44showringcleavagewithchargeretentionat the nonreducing terminus(see Scheme2b). The fragmention at m/z = 1272arisesfromloss of 1-octenefrom the n-octylp-aminobenzoategroup. (Reproducedwith permissionfrom Ref. 30.) Yn +a,-

CH=OH CHzOH "~"~O

O

+H

or

~

CHzOH • "'"~O

e.u~

+

Zrl ÷H

c.",oO"

.~ +H 1+~"

1,5 Xn c,,o,

.H 14-or-

_..j

C.H2OH

•

SCHEME2a. Reducingterminal ion series.

-

672

GLYCOCONJUGATES

[36]

Bn

c.,o.

~.,o.

CH2OH

CHzOH

1 '"

HO

H6

(~H ~ HO

OH

OH

J

Cn +or-

4. o1".

CH~'t

CH~OH

OHIO.

+H

CH~ +H or

"*"~0

0 HO

0/~'v''' -H

OH H/(~,I OH

~ O ~ O H HO OH

-H

+ HO OH

2,4 An CH~Z)H

CH2OH

C.H20H

C,HItOH

+H

0,2 An

_o%%

CH2OH

HO

OH

CH2OH

O

SCHEME 2b. Nonreducing terminal ion series.

Only one significant ion results from fragmentation of the derivatizing moiety itself, m/z 1272, from elimination of octene.

Asparagine-Linked Oligosaccharides The preponderance of work thus far has centered around structural elucidation of a variety of asparagine-linked ohgosaccharides, which may be liberated from natural and recombinant glycoproteins by treatment with Endo H or PNGaseF to yield glycosylation which is structurally homogeneous at their free reducing terminii. Thus, by reductive amination, microheterogeneity is immediately obvious from inspection of the LSIMS spectra in the positive ion mode in which little fragmentation usually occurs. Information regarding the class and type of antennae as well as

[36]

MS OF CHROMOPHORE-COUPLEDOLIGOSACCHARIDES

M a n v / ' ~ 9 8 0 656

453 [M-Hl1142

Man

M-Man 980 .

. '"",

673

.

MCLM-octene

.

I,

,,,,,,,'tt~'t'"~If'~,,,

.

,

~.............

,........

I

lIltl~lI~l~l~l~t~"~lII

1000

I

!',

1100

in

,

'I~'

1200

M-3 Man 656

M-2 Man

II

818 IIIII

.....................................................

ir''''~l

L.. , . . . . . . . . . .

,,.

IIIII

IIII

tl I III i

ii ILII

Iii

I,,I,ll',,l'rl'i''''~,,,~'ltl!t,,lurlI,Pl~Ill!,,,,,,,,l,,r'~lll!~'~ll,,,!l, 700

iii

~!l['

'800 '

900

M-3 Man-GIcNAc 453

460

soo

6oo

miz

FIG. 3. Negative ion LSIMS spectrum OfMan3GIcNAc~-ABOE.

information on branching of isobaric isomers may be obtained from the LSIMS spectra recorded in the negative ion mode. The negative ion LSIMS mass spectrum of the simplest branched member of this class is shown in Fig. 3.37 This trimannosyl structure with its chitobiose core intact was prepared as the octyl ester, and serves to

674

GLYCOCONJUGATES

[36]

illustrate the features of these spectra in the negative ion mode. Depending on the rigor with which the derivatized sample is desalted by washing with water on the reversed-phase HPLC, prior to initiation of the gradient, varying amounts of chloride attachment ions may be observed, such as the isotope cluster at m/z 1178, 1179, and 1180. Such chloride adduct ions in the negative ion mode or sodium adduct ions in the positive ion mode are relatively stable as compared with the corresponding molecular anions occurring for this oligosaccharide at m/z 1142. It can be readily seen that the most abundant fragments occurring in this spectrum are due to loss of a single anhydro mannosyl moiety at m/z 980, a triple mannosyl anhydro moiety at m/z 656, and cleavage of the chitobiose linkage with loss of Man3GlcNAc I anhydro moiety at m/z 453. In addition, there is a relatively minor loss corresponding to 2 residues of anhydromannose, m/z 818, thought to arise from double-cleavage processes which would be expected to be less abundant. 24 The negative ion mass spectrum of a Man6GlcNAc I isomer, prepared as the octyl ester and isolated from a yeast mnn mutant, is shown in Fig. 4a. It can readily be seen from the pattern of Y ions representing charge retention at the reducing terminal amino benzoate function that there is abundant loss of moieties resulting from simple elimination reactions corresponding to a monohexosyl, dihexosyl, a trihexosyl moiety, and, finally, a hexahexosyl moiety. Nonreducing A ions for Hex2 and Hex 3 m/z 383 and 585, respectively, are also present. The occurrence of these fragments taken together with the absence of significant ion current due to elimination of an analogous 4-mer or 5-mer establishes the branching pattern shown in structure I. For comparison, the mass spectrum of another Man6GlcNAc I carbohydrate, prepared as the ethyl ester, is shown M--.6M--.6M.--.nGN AC

t3 t3 M

M6~---M I

in Fig. 4b. This oligosaccharide was obtained from a gel filtration column fraction isolated from IgM, which was purified from the plasma of a patient with Waldenstrom's macroglobulinemia. 24 This fraction (d) was reductively aminated as the ethyl ester, and then subjected to reversedphase HPLC using an aminopropyl-bonded silica column as shown in Fig. 5. The major component d-3 was used to record the mass spectrum shown in Fig. 4b. From the general features of this spectrum, it can be seen that the pattern is similar to that shown in Fig. 4a, except for the shift of 84 mass units due to the mass difference between the octyl and ethyl ester derivatives. Except for the presence of additional, less abundant fragments at mass m/z 707 (Y4) and m/z 693 (A4), this spectrum supports structure

[36]

MS OF CHROMOPHORE-COUPLED OLIGOSACCHARIDES

675

(a)

(M-H)1425

(1) o

tO3 "ID t---1 e~

453




IL_ I

i

I

I

FIG. 5. HPLC separation of IgM-ABEE oligosaccharides: column fraction d. (Reproduced with permission from Ref. 24.)

676

GLYCOCONJUGATES

[36]

C

I

I

I

FIG. 6. HPLC separation of IgM-ABEE oligosaccharides: column fraction c. (Reproduced with permission from Ref. 24.)

1503 (M~)" 3U

545

~

TO7

1382

M5

FiG. 7. The spectrum obtained from fraction c-3. (G), Ions that arise from glycerol cluster ions. (Reproduced with permission from Ref. 24.)

II. The presence of these additional fragments indicates the presence of an additional branched isobar structure II!. The linkage differences Man

Man\ / 6 Man \

\ 6Man

Man /

\ ~Man--4GIcNAc--ABEE

Man--2Man

~Man--4GIcNAc--ABEE Man /

Man--2Man / II

III

between structures I and II have been established from consideration of other data presented in the original literature. 24,2sWhether information on linkage differences may be deduced from the mass spectra or CID spectra, in some cases, remains an open question. A further interesting example is that of a ManTGlcNAc] component, also from the immunoglobulin, which was treated in an analogous fashion, including HPLC separation, as shown in Fig. 6. The mass spectrum of HPLC fraction c-3 is shown in Fig. 7. From analogous interpretation of the negative ion mass spectrum, it can be seen that the major component is consistent with structure IV, but it is clear that there is at least two other possible branched isobars present consistent with structures V and VI. These results illustrate the quality of mass spectral information that

[36] Man

MS OF CHROMOPHORE=COUPLEDOLIGOSACCHARIDES \

Man--_,Man 6Man

Man/

N

~Man

Man /

\~Man--aGIcNAc--ABEE

Man--2 Man--_,Man/

677

Man--2Man

\ ~Man--4GIcNAc--ABEE

/

IV

V Man

\

Man--2Man /

6Man \~Man--4GIcNAc--ABEE

Man--2Man/ V!

can be obtained using this derivative in the negative ion mode in addition to the power of this approach in detecting the presence and partial structural nature of isobaric isomeric structures. While, in the published literature these components have not been separated, from recent Work in this laboratory using higher resolution chromatographic separation methodology, these branched isomers were separated into individual components such that the mass spectra of isomerically pure substances could be recorded. 37 To conclude this discussion of spectrum-structure correlations for the high mannose series, the fragmentation patterns of two Man9GicNAc~ isomers are presented in Figs. 8a and 8b to illustrate their qualitative differences. The mammalian Man9GlcNAc I shown in Fig. 8a was also obtained from IgM and corresponds to structure VII while the Man9 isomer shown in structure VIII was from an isolated m n n yeast mutant. Man--2Man 19)

(6)

~Man (4) ~ M'''~6M-'~6M'''*4G N Ac Man--2Man / ~Man--4GIcNAc--ABEE .],2 .],3 1,3 (to) (7) / M M M6*--M Man--2Man--2Man 1'2 (II) (8) (5) M VII ~,2 M VIII

In Fig. 8a it can be seen that in the Y series, fragments are observed for losses of 1-, 2-, 3-, 5-, and 9-mer moieties, whereas in Fig. 8b the most abundant Y series correspond to loss of I-, 2-, 4-, and 9-mer, respectively. A final example will indicate the ease of dealing with oligosaccharides of other classes, using an example from the complex class involving identification of the nature of oligosaccharides attached to recombinant hepatitis B pre-S 2 surface antigen. Reversed-phase HPLC of the oligosaccharides liberated by PNGase F were derivatized as the octyl ester as shown in

678

GLYCOCONJUGATES

1017

[36]

1341

i

7

I~

i

,.

.i

_

.t

+lo0

1821' (M-HI"

m/z

18IT

[M-HILIU -4 Hez 1141

1501

1|16

-2 Hex

-I Hez

l-

-3 Hez

m~

I

L.~

[

I|N

1600

mira

A

--

3111

'IOT 616

Ji £

'?

~,_ta ~ I . . . . . L . . L _ _ , IN

4 Hex

t

...........

-tl Heat . . l __.t . . . . . . . . . . . .

"

IN

-6 Herz 1 ....

+

mls

FIG. 8. (a) Negative ion LSIMS of fraction a-4 of the IgM-ABEE oligosaccharides. The spectrum showed an intense molecular anion at 1827. Series Y ions for losses of one, two, three or five mannoses were evident, as well as a less intense ion corresponding to the loss of four mannose residues (m/z 1179). Series 2,4,4 ions for fragments containing one, two, three, or five mannose residues were also observed. These data suggested that the branched residues in the molecule suppressed the formation of fragments consisting of six or seven mannose residues, evidence that the major component is formed from a triantennary core. (Reproduced with permission from Ref. 24.) (b) LSIMS o f m n n I mnn2 m n n l0 oligosaccharide MangGIcNAc. (Reproduced with permission from Ref. 28.)

[36]

MS OF CHROMOPHORE-COUPLEDOLIGOSACCHARIDES

679

REAGENT

NEt/IP,AL

320nm

m

f

FIG. 9. Reversed-phaseHPLC hepatitis B surface antigenprc-S2oligosaccharidefractionation primarily according to net charge. Fig. 9. Separation of fractions according to net charge is obtained. The fraction eluting as neutral oligosaccharides are shown in Fig. 10. The major component is a fucosylated biantennary structure IX, (M - H)- m/z 2019.

Gal

NAc

I0 / / Man--GIcNAc--GIcNAc--ABOE Gal__GlcNAc__Man/ 1873/ I Fuc \

IX

The presence of higher molecular weight additional neutral components are indicated by peaks at m/z 2384, representing a triantennary structure, and m/z 2165, representing a bifucosylated biantennary structure. Further, peaks at m/z 1857, 1653, and 1492 provide evidence for truncated structures present in this sample. 4°

Oligosaccharides Isolated from Nicotinic Acetylcholine Receptor Having established that n-alkyl p-aminobcnzoatcs with ester groups greater than ethyl could bc analyzed with higher sensitivityby LSIMS, wc incorporated this improvement into a simple, but effective,procedure 40B. L. GiUece-Castroand A. L. Burlingame,Proc. 36thAnnu. Conf. Mass Spectrom. Allied Topics, San Francisco, CA, p. 923 (1988).

680

GLYCOCONJUGATES

[36]

L,..I.I~ 1492

2019 1857

1653

j•iLA,jd 2165

~liJuL~eJ ,na---~:-~ !

uvl

6O0

1857 1653 J ~., Gal---GIcNAc---Man~ j

8O3

Gal---GIcNAc---Manj

1492

2384

803

600

(M-H)2109

Ma r ~ IcNAC----~IcNAc- ABOE / I 1873 Fuc

t

1330

f.

am

i

.

fW£

tIW

FIG. 10. Negative ion LSIMS of cloned hepatitis B pre-S2antigen oligosaccharides as the ABOE derivative.

for examining N-linked oligosaccharides released from microgram levels of several glycoproteins. We will present results obtained from studies of the nicotinic acetylcholine receptor (nAcChoR) as an example. In this case, the task was complicated by the size (270 kDa) of this multisubunit protein and its resistance to enzymatic digestion. In particular, the high levels of detergent, needed both to solubilize the protein and to aid digestion by PNGase F, had to be rigorously removed before any derivatization of the oligosaccharide or mass spectrometry could be attempted. Figure 11 shows the C~8 reversed-phase HPLC profile of the total oligosaccharide released from nAcChoR (250/xg, I nmol) and derivatized with n-hexyl p-aminobenzoate (no octyl ester of sufficient purity was available at the time of this study). As indicated above, reversed-phase chromatography separates these derivatives largely on the basis of the net charge carried, although shallower water/acetonitrile gradients can produce better resolution of species that carry the same charge but that differ in size. The profile obtained for the oligosaccharides from nAcChoR

[36]

MS OF CHROMOPHORE-COUPLEDOLIGOSACCHARIDES 0.1

.

681

,100

° :~ 0.05

.-~ 50 -~

15

30 Time/min

45

60

Fro. I I. C1~ reversed-phase HPLC profile of dcrivatized oligosaccharides from nAcChoR. (Reproduced with permission from Ref. 30.)

suggested the presence of small amounts of many acidic oligosaccharidcs, shown to be mono-, bi-, tri-, and tetrasialylated structures by mass spectrometry, as well as a large neutral fraction. LSIMS of the neutral fraction (20% of total) in both positive and negative ion modes afforded the spectra shown in Fig. 12a and 12b, respectively, revealing the components as high mannose oligosaccharides of composition ManaGlcNAc2 and MangGlcNAc2 . No molecular ions suggesting the presence of other high mannose species, hybrid or complex glycans were observed in this fraction. In addition to the singly and doubly charged molecular ions seen in the positive ion spectrum, three series of reducing terminal fragment ions, although of low abundance, were also apparent (see Scheme IIa,b for the nomenclature used). The intensity of the Y series of fragment ions was much greater in the negative ion mode (see Fig. 12b), clearly showing the losses of up to 4 and 5 hexose (mannose) units from MansGlcNAc 2 and MangGlcNAc2, respectively. Further purification of this fraction by HPLC using an amine-bonded column allowed LSIMS data to be obtained on each component. These spectra (data not shown) illustrated that the fragment ion at m/z = 1438 resulted from the loss of 3 hexose (mannose) units from MansGlcNAc2. This ion was not found in the spectrum of MangGlcNAc2 indicating that this oligosaccharide cannot lose 4 hexose (mannose) units by the cleavage of only one bond. The Y ion at m/z = 1114 in the negative ion LSIMS spectrum of the high mannose fraction is barely detected above the level of the chemical noise. This may be explained by the presence of a minor isomeric component, or may arise from the cleavage of two bonds of a major component structure. 41The structures derived from the mass spectral data were consistent 41 A. Dell and C. E. Ballou, Carbohydr. Res. 120, 95 (1983).

682

GLYCOCONJUGATES o

[36] 1926

MH +

MH+2088 Y

Y

l'5x

1440,.-+

z ~ ~Yr"

.

.

.

.

.

.

.

.

.

.

.

.

15 "r'"

1602 " ' +

z ] l,vK ? .[

" .........

""'

Y

• ~

, 1,5 x

~/o,4,,

+

z 1 ,,,,vK

MK* MN+

+klg÷

~

7 :?~..g_ ._..?:.~_, _"2_ ~;"Y"2...Ilk Ilill

MH ++ 2 M H +2+

Y L~X 1278/w

...J

Z. I

i"hti'~1I'h~i'~'~'h'~iii~d~qi1~hi~diiii~dtiiii~di~f''''d''''~i''hi~"'~hi~''''~i~'~'''"hii~Hi~:~iiH~dii~i~idi~"~d It

III

!~

......... 1,1,1,11111........ d.lll.,.,l

b (M-H~- 1924 Y 1438

y 1600 [

Y 1762 I

Zl

Z|

(M-H)- 2086 M.o-~,,],a! .... , ~ t

iiii

i

M*O*

1840 iiiii

FIG. 12. (a) Positive ion LSIMS mass Spectrum of MansGIcNAc2-ABHE and Man9GIcNAc2-ABHE from nAcChoR. Very weak reducing terminal fragment ions are observed (see Scheme 2a for nomenclature). Ions marked with an asterisk are matrix adducts involving the addition of a dehydrated glycerol molecule. (b) Negative ion LSIMS mass spectrum of MansGlcNAc2-ABHE and MangGlcNAc2-ABHE from nAcChoR. An abundant series of reducing terminal fragment ions (type Y) are observed (see Scheme 2a for nomenclature). The fragment ion at m/z = 1840 results from the loss of 1-hexene from the n-hexyl paminobenzoate group. (Reproduced with permission from Ref. 30.)

[36]

MS OF CHROMOPHORE-COUPLED OLIGOSACCHARIDES

with those obtained by the 1H NMR work of Nomoto shown as structures X and Xi.

e t al. 42

683

and are

(M - H)- 1924 1276

Mana{i-2)Man,x{l-6)\

Man,~{I-3)/ Man,~{1-6}~ / / Man~{I.4)GIcNAcl]{I-4)GlcNAc--ABHE Mana [ ~ M a n a I°2}Man'~'"3'~ 1438 1762

1600

X (M - H)-

Man,,tl-2)Man,~{i-6)\ Manc,{i.2)Mana{J.3)/

2086

1276

Man,~{l-6)/ YxMan~tl-4)GlcNAc~{1-4}GlcNAc--ABHE

Man~ [~Man'~ [ ~ MRR"{I-3~" 160o 1924

1762

Xl In the case of the whole nAcChoR, however, the total amount o f complex oligosaccharide present is low (