[29]
O V E R V I E W A N D STRATEGY
539
[29] G l y c o c o n j u g a t e s : O v e r v i e w a n d S t r a t e g y
By ROGER A. LAINE The chapters in this section reflect the latest and most useful methods in application of mass spectrometry to carbohydrates from biological samples. Tools for interpreting the data include example and standard spectra, and details of fragmentation mechanisms and pathways. This section should provide a good working knowledge in the use of mass spectrometry in structure analysis of carbohydrates, although, it is recommended that the primary references be consulted. In the interest of quoting the earliest relevant reference: "Teach him what has been said in the past; then he will set a good example . . . and judgement and all exactitude shall enter into him. Speak to him, for there is none born wise." 1 In this introduction there will be no attempt to review the background associated with the methods in all of the chapters. Instead I will point to an earlier historical perspective and address some practical aspects of carbohydrate analysis. The practical application of mass spectrometric techniques to the difficult task of complete structural elucidation of oligosaccharides, polysaccharides, and glycoconjugates has been influenced strongly by instrument development, namely, gas-liquid chromatography/mass spectrometry, soft ionization, and high-field magnets. First, methylation linkage analysis developed principally because of the first working interface between a gas-liquid chromatograph and a mass spectrometer (GC/MS) which was assembled by Ragnar Ryhage at the Karolinska Institute in Stockholm2on what would become the LKB-9000 instrument. The two-stage molecular separator, which constituted this interface, was ingenious and enabled online mass spectrometric analysis of gas-liquid chromatography effluents. The synergy of an on-line chromatographic and mass spectrometric system was astounding and still constitutes one of the most powerful analytical tools for volatiles. At about the same time, around 1964, Hakomori adapted Cory's methylsulfinyl carbanion reagent to efficiently and completely methylate sugi Ptahhotpe, "The Maxims of Ptahhotpe," Introduction. (2350 B.C.) 2 R. Ryhage, Anal. Chem. 36, 759 (1964).
METHODS IN ENZYMOLOGY, VOL. 193
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
540
GLYCOCONJUGATES
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ars. 3 Stimulated by these two developments and faced with difficulties of elucidating bacterial polysaccharides, Bengt Lindberg and colleagues at the University of Stockholm developed methods for separation of partially methylated alditol acetates by gas-liquid chromatography and identification of the linkage positions by electron ionization (EI)-MS 4 (reviewed in [30], this volume). This technology enabled rapid linkage position analysis on samples of 0.1 to 1 /zmol, and with a few improvements, is still the method most used for determination of linkage. Thus, we included the methodology for this crucial technique? Although paper, thin-layer, and gas chromatographic analysis of methylated sugars predated this technology, larger quantities of sample were required for those techniques, and characterized standards were necessary. Much later, improvements in the methods included fused quartz capillary gas chromatography, which cut by one-third the time necessary for gas-liquid chromatography separations. 4 Chemical ionization mass spectrometry added tenfold to the sensitivity,5 and a few improvements in the methods for methylation have been published. 6 However, the technique has remained fairly standard since 1970, practiced in only a handful of laboratories throughout the world. Fragmentation schemes for derivatized oligosaccharides were intensely studied in the years prior to 1975, using trimethylsilyl, methyl, and acetyl derivatives for direct evaporation and GC/MS applications. Principal researchers in this field were Biemann, DeJongh, and Schnoes, 7 and Chizhov and Kochetkov of the USSR, the latter having written early reviews on this subject, a'9 The Baltic seemed to connect researchers in the area of oligosaccharide and glycoconjugate mass spectrometry, including Karlsson ~° of Sweden, and Karkkainen from Finland. The latter showed that these compounds could be separated on a gas-liquid chromatograph. All of this work was extensively and thoroughly reviewed in 1974 by Lonngren and Svensson. H This review provides a valuable text for those who intend to acquire a background in mass spectrometry related to carbohydrate chemistry and provides a surprisingly rich lesson for those who believe that carbohydrate mass spectrometry began with fast atom bombardment ionization (FAB)-MS methods. Identification of nearly all 3 S.-I. Hakomori, J. Biochem. 55, 205 (1964). 4 C. G. Hellerqvist, this volume [30]. s R. A. Laine, Anal. Biochem. 116, 383 (1981). 6 I. Ciucanu and F. Kerek, Carbohydr. Res. 131, 209 (1984). 7 K. Biemann, D. C. DeJongh, and H. K. Schnoes, J. Am. Chem. Soc. 85, 1763 (1963). 8 0 . S. Chizhov and N. K. Kochetkov, Adv. Carbohydr. Chem. 21, 29 (1966). 9 0 . S. Chizhov and N. K. Kochetkov, Methods Carbohydr. Chem. 6, 540 (1972). 10 B. E. Samuelsson, W. Pimlott, and K.-A. Karlsson, this volume [34]. ii j. Lonngren and S. Svennson, Adv. Carbohydr. Chem. Biochem. 29, 42 (1974).
[29]
OVERVIEW AND STRATEGY
541
of the principal types of fragmentation in monosaccharides and oligosaccharides was established before 1974 as reviewed and described by Lonngren and Svennson.11 Surprisingly, few modern authors give proper credit to this large body of previous work. Another significant advance mentioned above was the success in mass spectrometry of derivatized oligosaccharides and glycoconjugates. Many of the results from which FAB-MS of carbohydrates takes root are based on the early work of K.-A. Karlsson I° and colleagues. EI spectra of very large permethylated,12 intact, and amide-reduced glycosphingolipids were first reported as early as 1973, and are nearly as informative as any FABMS spectra published today. 10Another early method with enduring qualities is the combination of high-temperature gas-liquid chromatography with mass spectrometry. Soft ionization applications of this technique for oligosaccharide measurements are described elsewhere in this volume.13 Since 100 nmol were often used for analysis by the EI methods, the third major impact on carbohydrate analysis has been the improvement in sensitivity using the new soft ionization methods related to FAB-MS. FAB-MS produces stable spectra with an intense molecular ion and an increase in sensitivity of 10- to 100-fold) 4'15 Array detectors increase the molecular ion detectability by another astonishing two orders of magnitude, yielding picomole sensitivity.16 A review of FAB-MS development, which accelerated progress regarding all polar biological compounds, is included in several of the chapters in this volume and need not be repeated here. The pioneering work by Kotchetkov, Chizov, and Karlsson as mentioned above has been extended to larger compounds by DeU,14 PeterKatalini6 and Egge, 15 Costello, 17 and Burlingame and colleagues j6'18 and many others too numerous to mention here. Novel lipoidal derivatives at the reducing end of oligosaccharides have made them more amenable to FAB ionization. 18 FAB is also used to map the location of carbohydrates on the protein chain. 19 Collision-induced dissociation (CID), when combined with FAB-MS to generate the molecular ion, gives a significant advance in assigning some of the many details of glycoconjugate structure. Early work in this i: K.-A. Karlsson, FEBS Lett. 32, 317 (1973). t3 G. C. Hansson and H. Karlsson, this volume [39]. 14 A. Dell, this volume [35]. t5 j. Peter-Katalini~ and H. Egge, this volume [38]. 16 B. L. GiUece-Castro and A. L. Burlingame, this volume [37]. 17 C. E. Costello and J. E. Vath, this volume [40]. 18 A. L. Burlingame and L. Poulter, this volume [36]. 19 S. A. GAIT,J. R. BAIT,G. D. Roberts, K. R. Anumula, and P. B. Taylor, this volume [27].
542
GLYCOCONJUGATES
[29]
area has been reviewed in several recent publications and the preambles of relevant chapters in this v o l u m e 16'17'2°'21'22 and need not be repeated here. High-energy collision (magnetic sector instruments) and low-energy collision (triple quadrupole instruments) may generate somewhat different sets of daughter ions. Costello's systematic nomenclature of the CID daughter ions 17is supplanting the older assignments reviewed by Lonngren and Svensson, 11and is also used by Gillece-Castro and Burlingame in this volume. 16Statistical treatment of glycosidic cleavages based on likelihood of cleavage of more hindered bonds (which may not easily dissipate collision energy) has been suggested by Laine et al. and supported by molecular modeling.2°'22A few other investigations have suggested that linkage information may be directly obtained from F A B - M S . 23-27 A particularly complete study of the use of FAB-MS and linked scanning for determination of linkage position of terminal saccharides in a set of 19 oligosaccharides was recently published by Garozzo e t al. ~8 A detailed interpretation of a number of CID glycoconjugate spectra based on traditional ion fragment-cleavage mechanisms is described elsewhere in this volume. 16,17Richter shows that epimer forms of sugars can be discerned in reducing terminal groups by CID methods. 21,29 Californium-252 fission-fragment plasma desorption time-of-flight (TOF) mass spectrometry (PDMS) has also been developed for complex carbohydrates, principally by Jardine in collaboration with Brennan. 3° Figure 1, taken from Jardine e t al., 3° gives an example of the spectral appearance and type of information available from PDMS. As currently practiced, PDMS only has mass resolution of about 0. I% and therefore at mass 1000, the accuracy is only within about 3 mass units. In practice, centroids of the isotope peak envelope can usually be estimated within one mass unit at mass 1000 and within 2 u at mass 2000. 20 R. A. Laine, this series, Vol. 179, p. 157. 21 W. J. Richter, D. R. Mfiller, and B. Domon, this volume [33]. 22 R. A. Laine, K. M. Pamidimukkala, A. D. French, R. W. Hall, S. A. Ahbas, R. K. Jain, and K. L. Matta, J. Am. Chem. Soc. 110, 6931 (1988). 23 y . Chen, N. Chen, M. Li, F. Zhao, and N. Chen, Biomed. Mass Spectrom. 14, 9 (1987). 24 j. p. Kamerling, W. Heerma, F. G. Vliegenthart, N. B. Green, I. A. S. Lewis, G. Strecker, and G. Spik, Biomed. Mass Spectrom. 10, 420 (1983). 25 Z. Lam, M. B. Comisarow, G. G. S. Dutton, D. A. Weil, and A. Biarnson, RapidCommun. Mass Spectrom. 1, 83 0987). 26 j. C. Prome, M. Aurelle, D. Prome, and D. Savagnac, Org. Mass Spectrom. 22, 6 (1987). 27 Z. Lam, M. B. Comisarow, and G. G. S. Dutton, Anal. Chem. 60, 2304 (1988). 28 D. Garozzo, K. M. Giuffrida, G. Impallomeni, A. Bailistreri, and G. Montaudo, Anal. Chem. 62, 279 (1990). 29 D. R. Muller, B. Doman, and W. J. Richter, Adv. Mass Spectrom. I1B (1989). 3o I. Jardine, G. Scanlan, M. NcNeil, and P. J. Brennan, Anal. Chem. 61, 416 (1989).
[29]
OVERVIEW AND STRATEGY
543
o i
R=
16000(
-CO(CI-~=sCH~
or -CO(CH=)t=CH= or - C O ( ~
331
=oos
1074
M. MALMOENSE PERACETYLATED ~ 8000( _=
MNa+
620 85O
3O32
m/z
FIG. 1. Positive ion 252Cffission fragment ionization time-of-flight (TOF) mass spectrum (plasma desorption mass spectrometry, PDMS) ofperacetylated Mycobacterium malmoense lipooligosaccharides (LOS). Recorded on a BIN-10K PDMS (Bio-Ion Nordic, Uppsala, Sweden) using accelerating voltage of 20 kV. LOS (3 /~g) was coated on nitrocelluloselayered aluminized Mylar foils. Spectra were acquired for 1 hr. (Reproduced from Ref. 30 with permission of the authors.)
This approach has been extensively used in peptide analysis. 31 Although mass spectra of peptides of 34,000 have been recorded with this technique, successful high-molecular weight analyses are rare and often dependent on double or triply charged ions. For peptides, in practice, masses below 6000 Da are often attainable and masses between 1000 and 3000 Da routine. McNeal e t al. 32 have used PDMS with ion-pairing reagents to examine sulfated oligosaccharides. We have also examined the usefulness of PDMS for saccharides of different types and derivatives, and have found a limit of DP (degree of polymerization) 13 for an underivatized dextran series.33 Upon acetylation, the upper limit under established conditions appears to 31 p. Roepstorff, this volume [23]. 32 C. J. McNeal, R. D. Macfarlane, and I. Jardine, Biochem. Biophys. Res. Coramun. 139, 18 (1986). 33 C. M. David and R. A. Laine, unpublished (1990).
544
GLYCOCONJUGATES
[29]
500
~q n 17-r~r II 18-mer
~ •
.
~
~
~
~/
II.
to
,~ i ~ = o
it Ii "¢
~25C
2500
3500
4500
5500
21- m e r
20-met 'z/
.....
65()0
M/Z FIG. 2. Positive ion 2nCf fission fragment ionization time-of-flight (TOF) mass spectrum
(plasma desorption mass spectrometry, PDMS) of peracetylated Pharmacia T-10 dextran oligomers fractionated by gel permeation chromatography. Spectra were recorded on a BioIon Nordic BIN-20 PDMS using accelerating voltage of 18 kV. Sample (1/~g) was coated on nitrocellulose-layered aluminized Mylar foils. Spectra were acquired for 1 hr.
be in the range ofm/z 6200 for MH ÷ and (MH-60) ÷ ions at a DP of 21 as shown in Fig. 2. 33 Chitooligomers gave good results up to DP 12, where solubility became a problem. Acetylation did not improve the performance for chitooligomers.33 The technique was obviously useful for the mycobacterial glycoconjugates 3° and heparin oligosaccharides,32 and may be a valid alternative for FAB-MS in molecular weight determination. N-Linked glycopeptides in the mass range 7000 to 12,000 Da, are found on glycoproteins such as human erythrocyte band 334 and human placental glycoproteins? 5 No one has yet defined an ionization technique that can show molecular weights or polymer distributions for carbohydrates of this mass. Matrix-assisted laser desorption TOF mass spectrometry may be one possibility to explore. Also, electrospray ionization may be possible J. Jarnefelt, J. Rush, Y.-T. Li, and R. A. Laine, J. Biol. Chem./,53, 8006 (1978). 35 B. C.-R. Zhu, S. F. Fisher, H. Pande, J. Calaycay, J. E. Shively, and R. A. Laine, J. Biol. Chem. ?,59, 3962 (1984).
[29]
OVERVIEW AND STRATEGY
545
if the N-acyl groups are removed from the carbohydrate, creating multiply charged sites. The reader will note there is a bit of the skills of the advertiser applied in each of the articles. Although there are a few pertinent caveats sprinkled in the chapters, each author is at least slightly optimistic about the extent of usefulness of his particular method. 36 "Naoita de oentis, de tauris nattat arator, enumerat miles vulnera, pastor ooes." 37Thus, nothing has changed in four millenia with regard to the value I and the prejudice37 of experts transmitting and extolling their experiences. In Section III of this volume you will find a selection of the world's specialists in carbohydrate analysis using mass spectrometry, each with a favorite approach. None, however, can be taken alone. Combinations of more than one method described here are necessary and synergistic. The complete elucidation of the structure of a complex carbohydrate requires analytical input from a number of methods, including wet chemical, enzymatic, antibody or lectin affinity, radiochromatography, thinlayer, paper, gas-liquid, or high-performance liquid chromatography. Sugar composition, ring size, anomeric configuration, position of linkage, internal sequence, branching, and noncarbohydrate substitution cannot all be gained from using one technique, although high-field two-dimensional (2D) NMR comes the closest. For pure samples in the range of 5 /zmol containing a reasonable number (fewer than 10) of monosaccharide units, NMR can provide complete structural data using a combination of two or more two-dimensional techniques including relay COSY, NOESY, HOHAHA, or TOCSY and other pulse sequence methods. Since NMR is nondestructive, and, as a lower limit (at least currently), ifa quantity more than 50 nmol is available, NMR spectra should be recorded on the sample before any destructive analytical procedure is used. Most problems in carbohydrate analysis arise not from synthesis nor from large-scale preparations, but from investigations of biologically active binding-recognition systems. Specificity usually dictates the existence of a rare sequence and, usually only picomole to nanomole quantities are readily available. Mass spectrometry finds its niche in high sensitivity studies. In Section III the reader will find the work of many investigators who apply mass spectrometry to nanomole to picomole amounts of complex carbohydrates. The hard fact is, however, that mass spectrometry alone cannot 36 F. Bollum, " I take a craftsman's view of the practice of science and of the scientist." Personal communication (ca. 1977). 37 Propertius, "Elegies." Book ii (i), p. 43. (51 B.C.) Roughly, "The sailor recants his experiences of great storms, the farmer carries forth about oxen, the soldier talks about wounds and the shepherd discusses sheep."
546
GLYCOCONJUGATES
[29]
yet give a complete structure. It is part of the goal of this introduction and overview to point out the current practical limitations to the analytical methods. Another goal is to suggest strategies and approaches for nanomolar amounts of oligosaccharides that would employ mass spectrometry to lead to complete elucidation of a structure. Many investigators not quoted in this chapter have contributed significantly in this field. It is unrealistic to expect total characterization of intact structures from mass spectra. In the literature, as Biemann notes elsewhere in this volume, 38examples are often taken from known structures which are then "interpreted" according to the ions which happen to occur in the spectra and can be readily explained, often ignoring the unassigned ions. Mass spectra of complete unknowns may not be so simple to interpret. From the empirical descriptions of fragmentation accompanying mass spectra of oligosaccharides, it is readily seen that this is a science in its embryonic stages of development. Despite the extensive early literature, there still seems to be the element of surprise in the origin and mechanism of formation of some fragments. Predictability of fragment ions in spectra according to detailed structure of oligosaccharides is becoming more exact from collision-induced dissociation and tandem mass spectrometry,16'17.2°-29 but it is far from perfect. Example of an Approach to a Biological Carbohydrate The author's particular prejudice lies in the following approach to a total carbohydrate structure (oligosaccharide): "The road to resolution lies by doubt . . . . ,, 39 1. Gain strong evidence for purity. It is virtually impossible to begin with biological extracts and proceed easily to an absolutely pure sample. This derives from the many similar or related structures that are usually present in a biological system. 4° One must be assiduous about this aspect of the analysis. (a) Utilize at least three chromatographic systems, preferably at least one with a derivatized sample, permethylated or peracetylated. (See this series, Volumes 28, 83, 138, 179.) (b) Pay strict attention to composition analysis (methods are described in earlier volumes of this serie s, including Volume s 28, 83, 138, 179) for estimation of size, complexity, and purity. 2. If 50 nmol or more are available, at least obtain a one-dimensional IH NMR spectrum. 3s K. Biemann, this volume [18]. 39F. Quarles, "Emblems." Book iv (No. 2), Epig. 2 (1635). 40C. L. M. Stuits, C. C. Sweeley, and B. A. Macher, this series, volume179, p. 167. Note origins and subtle structural differencesamong 250 glycolipids.
[29]
OVERVIEW AND STRATEGY
547
3. Application of mass spectroscopy: (a) Use 1-20 nmol of the intact compound depending on sensitivity of the instrument, for FAB-MS. (b) Methylate 10-25 nmol (more is desirable). Use 1 or 2 nmol of the methylated derivative for FAB-MS.14 Process the remainder for partially methylated alditol acetates, 4 or alternatively use reductive cleavage4~and process samples by GC/MS, preferably chemical ionization (CI). 5 (c) Utilize Nilsson's technique 42 of periodate oxidation of the intact oligosaccharide, reduction, methylation (or a modification of this method using acetylation, 43 and FAB-MS or direct chemical ionization (DCI). CID may dramatically help this technique, but no report of its application is yet published. One may need 20-50 nmol due to the necessity of carrying the sample through several chemical steps. 4. Determine the remainder of your strategy. For example, you will not know the anomeric configurations or the relative location of linkage positions and anomeric configurations within the oligosaccharide. After following steps 1-3 above, the reader will have knowledge concerning: (a) A reasonable estimation of degree of purity, but not proof. (b) Which sugar(s) belong to nonreducing termini, ff the reader has a free oligosaccharide, it can be reduced with borohydride and the sugar located at the reducing end can be determined. If one methylates this sample, one will also obtain the linkage of the reducing end sugar. Otherwise one must do it the hard way. (c) Sugar composition and stoichiometry, which will give clues regarding purity. (d) Linkage positions and quantity of each sugar type in the molecule (very valuable in estimating purity), but not their relative order in the chain. (e) Whether a branched chain exists in the sample. (f) Whether any sugars have nonsaccharidic substitutions (FAB-MS of the intact saccharide plus FAB-MS of the methylated sampie). (g) Size (molecular weight). The investigator may have some probable knowledge regarding: (a) Location of branch, if any (by FAB-MS and CID cleavages). (b) Anomeric set if NMR has been performed, but not anomeric sequence order. (c) Reducing terminal if oligosaccharide has been reduced. (d) Some information from FAB-MS on the internal order of the sugars, i.e., if it is a heterooligosaccharide, e.g., methylpentose-hexose-N-acetylhexosamine-hexose . . . . The application of mass spectrometry will not provide the investigator with information Concerning the following: (a) Internal sugar order (man-
41 G. R. Gray, this volume [31]. 42 A.-S. Angel and B. Nilsson, this volume [32]. 43 C. G. Hellerqvist, personal communication (1990).
548
GLYCOCONJUGATES
[29]
nose-galactose-glucose) for oligomers where adjacent constituents have the same molecular weight, other than nonreducing terminal(s). (b) If identical sugars with different internal linkages exist, the internal order of the linkages will not be clear. However Nilsson's technique (this volume [32]43 may reveal some information regarding this aspect. (c) Anomeric configurations and their internal order among the constituent sugars. The following steps are necessary to establish the structure: 1. Determine the ring size: As pointed out by Gray in this volume, 41 the 4-substitution of a hexopyranose will give the same partially methylated alditol acetate as the 5-1inked furanose. Notwithstanding that 5-1inked furanoses are either extremely rare or nonexistent in most biological systems, we must prove the structure. Therefore, a few methods exist which can assist with this determination. The one most often used is sequential release of the terminal sugars by glycosylhydrolases (see other volumes in this series: Vols. 28, 83, 138, 179) specific for pyranoses after the terminal residues are identified as to their ring size by methylation linkage analysis. Testing a terminal sialic acid for anomeric linkage is relatively simple. Since biosynthesis has mainly used the tz bond in sialic acids, testing asialidases from one or more sources will usually result in a released sialic acid, revealing the penultimate sugar. When the enzymatically shortened oligosaccharide product is methylated, the ring form of the new nonreducing terminus sugar will be revealed by the Lindberg method. 4 Enzymes specific for this newly revealed terminal sugar can then be used to determine its anomeric specificity and ring size. Another glycosidase will reveal the next sugar, etc. Gray offers an alternative method41: reductive cleavage of the methylated oligosaccharide resulting in preservation of the ring form which can be identified by gas-liquid chromatography/EI-MS. 2. Anomeric set in the absence of NMR data: The most common microscale anomeric determinations are made by a- and fl-glycosylhydrolases when available. Once the sugar composition is known, the enzymes can be tested on the sample followed by chromatography to reveal mobility differences caused by loss of a sugar. As each successive saccharide is removed, a new nonreducing end is available for another exoglycosidase. Occasionally, endoglycosidases are useful. 34Methylation linkage analysis after each successive enzyme release will establish ring size and absolute sequence. Branching or tandem repeated identical residues complicate the problem. Two or more sugars may be removed at once. This raises the question: Were they connected in tandem or on different branches? Obviously, this must be examined by repetitive continuous analysis during enzyme treatment by FAB-MS, preferably with C I D . 16'17 Chemically, chromium trioxide oxidation of the peracetylated oligosac-
[29]
OVERVIEW AND STRATEGY
549
charide in glacial acetic acid has allowed discrimination between O~-D and/3-D bonds. 44 The fl-D-glycosidic linkages has two oxygens in close proximity, the ring and glycosidic oxygen which renders it more susceptible to oxidation by CrO3 than the a bonds. 44 Thus, sugar composition before and after oxidation will assign the survivors as a. Partial survivors suggest mixtures of a and/3 in the sample, either in the same saccharide or in a mixture of sacchafides. CID after chemical ionization was reported to discern among anomers in a complete set of glucose anomeric pairs of each linkage type, using mass-analyzed ion kinetic energy spectra (MIKES), 45 but this discovery has not been developed into a routine system. Solving Difficult Problems
Example I. An extract from a certain tissue may contain a group of 3 to l0 glycosphingolipids with a tetrasaccharide sugar chain, or a glycoprotein may yield as many as 30-60 different N-linked saccharides. Some of these may differ by only one position of linkage, an anomeric configuration or a branch. 4° A fast atom bombardment mass spectrum of such a mixture of several compounds which contain four sugars comprised of galactose, glucosamine, galactosamine, and glucose may be impossible to discern from that of a spectrum of a pure compound. Molecules of the approximate structure Gal-GalNAc-Gal-Glu-ceramide and Gal-GluNAc-Gal-Glu-ceramide may give identical spectra. In fact, they may give identical CID spectra. An example of a mixture consisting of branched and linear compounds includes Gal(fll--+3)
\ /
Gal(fl l--->4)Glu(fl1--*1)ceramide
GalNAc(fl 1---->4) I
Gal(fll--*4)
\
/
Gal(fl 1-*4)Glu(fl 1--*1)ceramide
GalNAc(fl 1--->3) II Gal(cd---)3)
\ /
Gal(fl 1---~4)Gluffl1---~1)ceramide
GalNAc(fl 1---->4) III 44 j. Hoffman, B. Lindberg, and S. Svensson, J. Supramol. Struct. Suppl. 1, 31 (1972). 45 E. G. de Jong, W. Heerma, and D. Dijkstra, Biomed. Mass Spectrom. 7, 127 (1980).
550
GLYCOCONJUGATES
[29]
Gal(fll---)3)GalNAc(fll----~4)Galffll---)4)Glu(fl1--*l)ceramide IV Gal(fl 1---)4)GalNAc(fl l---)4)Gal(fl1---*4)Gluffl1---~1)ceramide V
A mixture like this is difficult to resolve chromatographically and may give mass spectra from which one could draw one of several conclusions. All the members of this family of compounds have the same mass and relative polarity. In fact, they all have identical sugar compositions. The molecular ion may appear to lose a hexose or an N-acetylhexosamine as the nonreducing terminal moiety, or a mixture of the two losses may be seen. The linear compounds IV and V will produce fragments from cleavage of the terminal disaccharide while any of the pure compounds I, II, or III would show no cleavage of a terminal disaccharide, but would show fragments derived from a terminal trisaccharide. Nilsson's method 42 of periodate, permethylation, and FAB could not distinguish among I, II, and Ill, and would have some difficulty with IV because the penultimate GalNAc when linked by either its 3- or 4-position is resistant to periodate. His method is a valid third step, however, after determination of sugar composition, FAB-MS, and methylation linkage analysis. The investigator must be extremely vigilant concerning stoichiometry in analysis, and careful about using a number of different purification methods, both with intact and derivatized samples, utilizing several parallel methods before predicting structure. Dell has suggested one of the most powerful combinations in dealing with unknownsl4: Permethylate the compound; use a part for FAB-MS and a part for methylation linkage analysis. Example 2. Consider a pure compound of the structure: Neu5Ac(~t2---)3)Gal(fll---~4)GluNAc(fll---~4)Gal(fll---~4)Glu-ceramide VI
The strategy used will b e to establish the molecular weight and sugar/lipid composition by FAB-MS, giving a mass which is consistent with the sugar composition listed above and a C18-sphingenine attached to a Czs saturated fatty acid. The FAB spectrum may provide evidence that the sequence of sugar types is as shown above. If the structure were branched, the spectrum may show some differencesJ °,14-17 A methylation linkage analysis 4,5 will establish the composition of the linkage types and a partial structure of VI can be depicted as below: Neu5Ac(ct2--~ ?)Hex ?(fl 1--~4)GluNAc(fl 1--~?)Hex?(fl 1--,4)Hex?-ceramide
where Hex represents hexose. Obviously a large number of possible compounds still remain to be eliminated to find one structure for VI. The
[29]
OVERVIEW AND STRATEGY
551
questions remain because the FAB ionization mass spectrum cannot be used to distinguish among hexoses within the interior of the molecule, in this case o-galactose and o-glucose, although Richter's results look promising. 21,29 Hellerqvist notes 4 that one should take into account the known pathways of biosynthesis for each type of compound. For example, the most common structures in this class of compound have the glucose attached to the ceramide. Thus, a "likely" sequence would be as follows: Neu5Ac(~2---~ ?)Gal(fl l---~4)GluNAc(fl I---~?)Gal(fl 1-~4)Glu-ceramide
However, it should be remembered that if we a r e " completely characterizing" molecules, a "likely" structure is not an absolute one. Nilsson's method 42 helps greatly in this situation. The penultimate galactose will survive periodate oxidation, showing that it is the 3-1inked galactose, the GIcNAc will survive, but this could mean either a 3- or 4-1ink. (Prior hydrazinolysis here would give a free amino group at the 2 position of the GIcNAc. The 4-1inked GIcNAc would become periodate sensitive, rendering the Nilsson technique more useful.) The next galactose will be cleaved between carbons 2 and 3, showing by a specific set of fragments42 that this is the 4-1inked galactose, etc. The question marks remaining point to the fact that the molecule under study (VI) has both a 3- and a 4-1inked o-galactose. Without the Nilsson experiment, 42their order within the chain is not clear. Since the necessary sugar composition analysis shows 2 galactoses, a sequential enzyme degradation would eventually discriminate among the two possible arrangements of galactoses. Removal of the sialic acid followed by methylation would give a 4-1inked and a terminal galactose identifying the original location of the 3-1inked galactose. An exciting possibility is that a set of through-the-ring cleavages with CID and MS/MS might lead to the correct answer. 16.17 Example 3. There are some structures which defy ordinary methods of structural discernment, for example, the lactosamine oligomers shown below. Gal(fl 1---~4)GluNAc(fl 1--~6)
\
Gal(fll--~4)GluNAc(/31---~3)Gal(fll--~4)GluNAc(fll---~3)Gal(fl VII
l--~4)Glu-
Gal(fl l-~4)GluNAc(fl 1---~6)
\
Gal(fl 1--~4)GluNAc(/3 l--~3)Gal(fl 1--~4)GluNAc(fl l--~3)Gal(fl l--~4)GluVIII
Upon sequential methylation analysis and enzyme degradation, a common simple strategy for complete analysis, the products in both cases are identical. Hence, methylation analysis gives the identical result for both, yielding the following: Terminal galactose, 2 mol; 3-substituted galactose,
552
GLYCOCONJUGATES
[29]
1 mol; 3,6-disubstituted galactose, 1 mol; 4-substituted GIcNAc, 3 mol; and 4-substituted glucose, 1 mol. Sequential enzyme degradation with/3galactosidase and/3-hexosaminidase yields the following: galactose, 2 mol and GIcNAc, 2 mol. The fragment IX results from both structures: Gal(fl 1--*4)GluNAc(fll---~3)Gal(fl l---~4)Glu-ceramide iX
R e s u l t . The results of example 3 described above lead to the following conclusions: 1. No combination of methylation linkage analysis and exoglycosylhydrolases can resolve structures VII and VIII. 2. Nilsson's technique cannot resolve structures VII and VIII because the internal residues are all periodate-resistant.42 3. FAB-MS of any of the intact, permethylated or peracetylated compound should give a result which shows that compound VII gives a pentasaccharide B fragment17 while not easily yielding a tetrasaccharide B fragment. Compound VIH would give a tetrasaccharide B fragment and not a pentasaccharide B fragment. Some problems with yield of the fragment could occur because of the propensity for charge retention (inpositive ion FAB-MS) on the GIcNAc in the cleavage reactions such that compound VII could yield a lowabundance pentasaccharide fragment compared with B fragments from cleavages at the GIcNAc glycosidic bonds. A mixture of these two compounds in a sample would be a horrendous analytical enigma. Treating first with/3-galactosidase would give smaller compounds for mass spectrometry which would yield a trisaccharide fragment but not a disaccharide for VII while a disaccharide would be easily obtained from VIII. 46 CID of the fragment ions formed in either would answer the question. Solutions to other difficult carbohydrate analytical problems which may be encountered are given below along with pertinent references. 1. Location of substituents on cyclitols was resolved by Hsieh et al. 47 where an myo-inositol was substituted with both a phosphodiester and a glycosidically linked sugar. The linkages were characterized by a combination of periodate oxidation and analysis of the stereochemically distinct products by gas-liquid chromatography and chemical ionization mass spectrometry. 2. Location of sulfates on heterooligosaccharides was resolved for glycosaminoglycan oligosaccharides by Dell et al. 48 using substitution of 46 K. Watanabe, R. A. Laine, and S.-I. Hakomori, Biochemistry 14, 2725 (1975). 47 T. C.-Y. Hsieh, K. Kaul, R. A. Laine, and R. L. Lester, J. Biol. Chem. 253, 3575 (1978). A. Dell, M. E. Rogers, and J. E. Thomas-Oates, Carbohydr. Res. 179, 7 (1988).
[29]
OVERVIEW AND STRATEGY
553
the sulfates with acetates after permethylation under conditions which retained the sulfates. 3. Hellerqvist e t al. 49 located acyl groups by methyl vinyl ether acetalization5° of free .hydroxyls, an alkali-stable substitution, followed by methyl sulfinyl carbanion and methyl iodide treatment to replace acyl groups with methyl or other alkyl functions. 4. Quantitation of picomole to nanomole amounts of glycosidically bound monosaccharides in complex biological matrices have been attempted by a number of authors. Chemical ionization mass spectrometry was used with gas-liquid chromatography and computer-reconstructed mass chromatograms to analyze N-acetylneuraminic acid directly from erythrocytes by Ashraf e t al. 51 Roboz e t al. 52 and Sugawara e t al. 53 have used similar approaches. Sugiyama e t a l : 4 have also used GC/MS for estimation of free sialic acid. These methods could be adapted for any monosaccharide or partially methylated sugar) The problems of purity, stereochemical differences and identical mass and polarity in mixtures of oligosaccharides make mass spectrometry of this class of compounds most challenging. Still, one can see that enormous progress has been made in technology and ingenuity of approach. We can look forward to more sensitive instruments, novel energy-adding methods for the tandem mass spectrometric methods, and computer fingerprinting of data.
49 C. G. Hellerqvist, B. Lindberg, S. Svennson, and Lindberg, Carbohydr. Res. 8, 43 (1968). 5o A. N. DeBelder and B. Norrman, Carbohydr. Res. 8, 1 (1968). 51 j. Ashraf, D. A. Butterfield, J. Jarnefelt, and R. A. Laine, J. Lipid. Res. 21, 1137 (1980). 52 j. Roboz, R. Suzuki, and J. G. Bekesi, Anal. Biochem. 87, 195 (1978). 53 y . Sugawara, M. Iwamori, J. Portaukalian, and Y. Nagai, Anal. Biochem. 132, 147 (1983). 54 N. Sugiyama, K.-I. Saito, H. Mizu, M. Ito, and Y. Nagai, Anal. Biochem. 170, 140 (1988).
O V E R V I E W A N D STRATEGY
539
[29] G l y c o c o n j u g a t e s : O v e r v i e w a n d S t r a t e g y
By ROGER A. LAINE The chapters in this section reflect the latest and most useful methods in application of mass spectrometry to carbohydrates from biological samples. Tools for interpreting the data include example and standard spectra, and details of fragmentation mechanisms and pathways. This section should provide a good working knowledge in the use of mass spectrometry in structure analysis of carbohydrates, although, it is recommended that the primary references be consulted. In the interest of quoting the earliest relevant reference: "Teach him what has been said in the past; then he will set a good example . . . and judgement and all exactitude shall enter into him. Speak to him, for there is none born wise." 1 In this introduction there will be no attempt to review the background associated with the methods in all of the chapters. Instead I will point to an earlier historical perspective and address some practical aspects of carbohydrate analysis. The practical application of mass spectrometric techniques to the difficult task of complete structural elucidation of oligosaccharides, polysaccharides, and glycoconjugates has been influenced strongly by instrument development, namely, gas-liquid chromatography/mass spectrometry, soft ionization, and high-field magnets. First, methylation linkage analysis developed principally because of the first working interface between a gas-liquid chromatograph and a mass spectrometer (GC/MS) which was assembled by Ragnar Ryhage at the Karolinska Institute in Stockholm2on what would become the LKB-9000 instrument. The two-stage molecular separator, which constituted this interface, was ingenious and enabled online mass spectrometric analysis of gas-liquid chromatography effluents. The synergy of an on-line chromatographic and mass spectrometric system was astounding and still constitutes one of the most powerful analytical tools for volatiles. At about the same time, around 1964, Hakomori adapted Cory's methylsulfinyl carbanion reagent to efficiently and completely methylate sugi Ptahhotpe, "The Maxims of Ptahhotpe," Introduction. (2350 B.C.) 2 R. Ryhage, Anal. Chem. 36, 759 (1964).
METHODS IN ENZYMOLOGY, VOL. 193
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
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GLYCOCONJUGATES
[29]
ars. 3 Stimulated by these two developments and faced with difficulties of elucidating bacterial polysaccharides, Bengt Lindberg and colleagues at the University of Stockholm developed methods for separation of partially methylated alditol acetates by gas-liquid chromatography and identification of the linkage positions by electron ionization (EI)-MS 4 (reviewed in [30], this volume). This technology enabled rapid linkage position analysis on samples of 0.1 to 1 /zmol, and with a few improvements, is still the method most used for determination of linkage. Thus, we included the methodology for this crucial technique? Although paper, thin-layer, and gas chromatographic analysis of methylated sugars predated this technology, larger quantities of sample were required for those techniques, and characterized standards were necessary. Much later, improvements in the methods included fused quartz capillary gas chromatography, which cut by one-third the time necessary for gas-liquid chromatography separations. 4 Chemical ionization mass spectrometry added tenfold to the sensitivity,5 and a few improvements in the methods for methylation have been published. 6 However, the technique has remained fairly standard since 1970, practiced in only a handful of laboratories throughout the world. Fragmentation schemes for derivatized oligosaccharides were intensely studied in the years prior to 1975, using trimethylsilyl, methyl, and acetyl derivatives for direct evaporation and GC/MS applications. Principal researchers in this field were Biemann, DeJongh, and Schnoes, 7 and Chizhov and Kochetkov of the USSR, the latter having written early reviews on this subject, a'9 The Baltic seemed to connect researchers in the area of oligosaccharide and glycoconjugate mass spectrometry, including Karlsson ~° of Sweden, and Karkkainen from Finland. The latter showed that these compounds could be separated on a gas-liquid chromatograph. All of this work was extensively and thoroughly reviewed in 1974 by Lonngren and Svensson. H This review provides a valuable text for those who intend to acquire a background in mass spectrometry related to carbohydrate chemistry and provides a surprisingly rich lesson for those who believe that carbohydrate mass spectrometry began with fast atom bombardment ionization (FAB)-MS methods. Identification of nearly all 3 S.-I. Hakomori, J. Biochem. 55, 205 (1964). 4 C. G. Hellerqvist, this volume [30]. s R. A. Laine, Anal. Biochem. 116, 383 (1981). 6 I. Ciucanu and F. Kerek, Carbohydr. Res. 131, 209 (1984). 7 K. Biemann, D. C. DeJongh, and H. K. Schnoes, J. Am. Chem. Soc. 85, 1763 (1963). 8 0 . S. Chizhov and N. K. Kochetkov, Adv. Carbohydr. Chem. 21, 29 (1966). 9 0 . S. Chizhov and N. K. Kochetkov, Methods Carbohydr. Chem. 6, 540 (1972). 10 B. E. Samuelsson, W. Pimlott, and K.-A. Karlsson, this volume [34]. ii j. Lonngren and S. Svennson, Adv. Carbohydr. Chem. Biochem. 29, 42 (1974).
[29]
OVERVIEW AND STRATEGY
541
of the principal types of fragmentation in monosaccharides and oligosaccharides was established before 1974 as reviewed and described by Lonngren and Svennson.11 Surprisingly, few modern authors give proper credit to this large body of previous work. Another significant advance mentioned above was the success in mass spectrometry of derivatized oligosaccharides and glycoconjugates. Many of the results from which FAB-MS of carbohydrates takes root are based on the early work of K.-A. Karlsson I° and colleagues. EI spectra of very large permethylated,12 intact, and amide-reduced glycosphingolipids were first reported as early as 1973, and are nearly as informative as any FABMS spectra published today. 10Another early method with enduring qualities is the combination of high-temperature gas-liquid chromatography with mass spectrometry. Soft ionization applications of this technique for oligosaccharide measurements are described elsewhere in this volume.13 Since 100 nmol were often used for analysis by the EI methods, the third major impact on carbohydrate analysis has been the improvement in sensitivity using the new soft ionization methods related to FAB-MS. FAB-MS produces stable spectra with an intense molecular ion and an increase in sensitivity of 10- to 100-fold) 4'15 Array detectors increase the molecular ion detectability by another astonishing two orders of magnitude, yielding picomole sensitivity.16 A review of FAB-MS development, which accelerated progress regarding all polar biological compounds, is included in several of the chapters in this volume and need not be repeated here. The pioneering work by Kotchetkov, Chizov, and Karlsson as mentioned above has been extended to larger compounds by DeU,14 PeterKatalini6 and Egge, 15 Costello, 17 and Burlingame and colleagues j6'18 and many others too numerous to mention here. Novel lipoidal derivatives at the reducing end of oligosaccharides have made them more amenable to FAB ionization. 18 FAB is also used to map the location of carbohydrates on the protein chain. 19 Collision-induced dissociation (CID), when combined with FAB-MS to generate the molecular ion, gives a significant advance in assigning some of the many details of glycoconjugate structure. Early work in this i: K.-A. Karlsson, FEBS Lett. 32, 317 (1973). t3 G. C. Hansson and H. Karlsson, this volume [39]. 14 A. Dell, this volume [35]. t5 j. Peter-Katalini~ and H. Egge, this volume [38]. 16 B. L. GiUece-Castro and A. L. Burlingame, this volume [37]. 17 C. E. Costello and J. E. Vath, this volume [40]. 18 A. L. Burlingame and L. Poulter, this volume [36]. 19 S. A. GAIT,J. R. BAIT,G. D. Roberts, K. R. Anumula, and P. B. Taylor, this volume [27].
542
GLYCOCONJUGATES
[29]
area has been reviewed in several recent publications and the preambles of relevant chapters in this v o l u m e 16'17'2°'21'22 and need not be repeated here. High-energy collision (magnetic sector instruments) and low-energy collision (triple quadrupole instruments) may generate somewhat different sets of daughter ions. Costello's systematic nomenclature of the CID daughter ions 17is supplanting the older assignments reviewed by Lonngren and Svensson, 11and is also used by Gillece-Castro and Burlingame in this volume. 16Statistical treatment of glycosidic cleavages based on likelihood of cleavage of more hindered bonds (which may not easily dissipate collision energy) has been suggested by Laine et al. and supported by molecular modeling.2°'22A few other investigations have suggested that linkage information may be directly obtained from F A B - M S . 23-27 A particularly complete study of the use of FAB-MS and linked scanning for determination of linkage position of terminal saccharides in a set of 19 oligosaccharides was recently published by Garozzo e t al. ~8 A detailed interpretation of a number of CID glycoconjugate spectra based on traditional ion fragment-cleavage mechanisms is described elsewhere in this volume. 16,17Richter shows that epimer forms of sugars can be discerned in reducing terminal groups by CID methods. 21,29 Californium-252 fission-fragment plasma desorption time-of-flight (TOF) mass spectrometry (PDMS) has also been developed for complex carbohydrates, principally by Jardine in collaboration with Brennan. 3° Figure 1, taken from Jardine e t al., 3° gives an example of the spectral appearance and type of information available from PDMS. As currently practiced, PDMS only has mass resolution of about 0. I% and therefore at mass 1000, the accuracy is only within about 3 mass units. In practice, centroids of the isotope peak envelope can usually be estimated within one mass unit at mass 1000 and within 2 u at mass 2000. 20 R. A. Laine, this series, Vol. 179, p. 157. 21 W. J. Richter, D. R. Mfiller, and B. Domon, this volume [33]. 22 R. A. Laine, K. M. Pamidimukkala, A. D. French, R. W. Hall, S. A. Ahbas, R. K. Jain, and K. L. Matta, J. Am. Chem. Soc. 110, 6931 (1988). 23 y . Chen, N. Chen, M. Li, F. Zhao, and N. Chen, Biomed. Mass Spectrom. 14, 9 (1987). 24 j. p. Kamerling, W. Heerma, F. G. Vliegenthart, N. B. Green, I. A. S. Lewis, G. Strecker, and G. Spik, Biomed. Mass Spectrom. 10, 420 (1983). 25 Z. Lam, M. B. Comisarow, G. G. S. Dutton, D. A. Weil, and A. Biarnson, RapidCommun. Mass Spectrom. 1, 83 0987). 26 j. C. Prome, M. Aurelle, D. Prome, and D. Savagnac, Org. Mass Spectrom. 22, 6 (1987). 27 Z. Lam, M. B. Comisarow, and G. G. S. Dutton, Anal. Chem. 60, 2304 (1988). 28 D. Garozzo, K. M. Giuffrida, G. Impallomeni, A. Bailistreri, and G. Montaudo, Anal. Chem. 62, 279 (1990). 29 D. R. Muller, B. Doman, and W. J. Richter, Adv. Mass Spectrom. I1B (1989). 3o I. Jardine, G. Scanlan, M. NcNeil, and P. J. Brennan, Anal. Chem. 61, 416 (1989).
[29]
OVERVIEW AND STRATEGY
543
o i
R=
16000(
-CO(CI-~=sCH~
or -CO(CH=)t=CH= or - C O ( ~
331
=oos
1074
M. MALMOENSE PERACETYLATED ~ 8000( _=
MNa+
620 85O
3O32
m/z
FIG. 1. Positive ion 252Cffission fragment ionization time-of-flight (TOF) mass spectrum (plasma desorption mass spectrometry, PDMS) ofperacetylated Mycobacterium malmoense lipooligosaccharides (LOS). Recorded on a BIN-10K PDMS (Bio-Ion Nordic, Uppsala, Sweden) using accelerating voltage of 20 kV. LOS (3 /~g) was coated on nitrocelluloselayered aluminized Mylar foils. Spectra were acquired for 1 hr. (Reproduced from Ref. 30 with permission of the authors.)
This approach has been extensively used in peptide analysis. 31 Although mass spectra of peptides of 34,000 have been recorded with this technique, successful high-molecular weight analyses are rare and often dependent on double or triply charged ions. For peptides, in practice, masses below 6000 Da are often attainable and masses between 1000 and 3000 Da routine. McNeal e t al. 32 have used PDMS with ion-pairing reagents to examine sulfated oligosaccharides. We have also examined the usefulness of PDMS for saccharides of different types and derivatives, and have found a limit of DP (degree of polymerization) 13 for an underivatized dextran series.33 Upon acetylation, the upper limit under established conditions appears to 31 p. Roepstorff, this volume [23]. 32 C. J. McNeal, R. D. Macfarlane, and I. Jardine, Biochem. Biophys. Res. Coramun. 139, 18 (1986). 33 C. M. David and R. A. Laine, unpublished (1990).
544
GLYCOCONJUGATES
[29]
500
~q n 17-r~r II 18-mer
~ •
.
~
~
~
~/
II.
to
,~ i ~ = o
it Ii "¢
~25C
2500
3500
4500
5500
21- m e r
20-met 'z/
.....
65()0
M/Z FIG. 2. Positive ion 2nCf fission fragment ionization time-of-flight (TOF) mass spectrum
(plasma desorption mass spectrometry, PDMS) of peracetylated Pharmacia T-10 dextran oligomers fractionated by gel permeation chromatography. Spectra were recorded on a BioIon Nordic BIN-20 PDMS using accelerating voltage of 18 kV. Sample (1/~g) was coated on nitrocellulose-layered aluminized Mylar foils. Spectra were acquired for 1 hr.
be in the range ofm/z 6200 for MH ÷ and (MH-60) ÷ ions at a DP of 21 as shown in Fig. 2. 33 Chitooligomers gave good results up to DP 12, where solubility became a problem. Acetylation did not improve the performance for chitooligomers.33 The technique was obviously useful for the mycobacterial glycoconjugates 3° and heparin oligosaccharides,32 and may be a valid alternative for FAB-MS in molecular weight determination. N-Linked glycopeptides in the mass range 7000 to 12,000 Da, are found on glycoproteins such as human erythrocyte band 334 and human placental glycoproteins? 5 No one has yet defined an ionization technique that can show molecular weights or polymer distributions for carbohydrates of this mass. Matrix-assisted laser desorption TOF mass spectrometry may be one possibility to explore. Also, electrospray ionization may be possible J. Jarnefelt, J. Rush, Y.-T. Li, and R. A. Laine, J. Biol. Chem./,53, 8006 (1978). 35 B. C.-R. Zhu, S. F. Fisher, H. Pande, J. Calaycay, J. E. Shively, and R. A. Laine, J. Biol. Chem. ?,59, 3962 (1984).
[29]
OVERVIEW AND STRATEGY
545
if the N-acyl groups are removed from the carbohydrate, creating multiply charged sites. The reader will note there is a bit of the skills of the advertiser applied in each of the articles. Although there are a few pertinent caveats sprinkled in the chapters, each author is at least slightly optimistic about the extent of usefulness of his particular method. 36 "Naoita de oentis, de tauris nattat arator, enumerat miles vulnera, pastor ooes." 37Thus, nothing has changed in four millenia with regard to the value I and the prejudice37 of experts transmitting and extolling their experiences. In Section III of this volume you will find a selection of the world's specialists in carbohydrate analysis using mass spectrometry, each with a favorite approach. None, however, can be taken alone. Combinations of more than one method described here are necessary and synergistic. The complete elucidation of the structure of a complex carbohydrate requires analytical input from a number of methods, including wet chemical, enzymatic, antibody or lectin affinity, radiochromatography, thinlayer, paper, gas-liquid, or high-performance liquid chromatography. Sugar composition, ring size, anomeric configuration, position of linkage, internal sequence, branching, and noncarbohydrate substitution cannot all be gained from using one technique, although high-field two-dimensional (2D) NMR comes the closest. For pure samples in the range of 5 /zmol containing a reasonable number (fewer than 10) of monosaccharide units, NMR can provide complete structural data using a combination of two or more two-dimensional techniques including relay COSY, NOESY, HOHAHA, or TOCSY and other pulse sequence methods. Since NMR is nondestructive, and, as a lower limit (at least currently), ifa quantity more than 50 nmol is available, NMR spectra should be recorded on the sample before any destructive analytical procedure is used. Most problems in carbohydrate analysis arise not from synthesis nor from large-scale preparations, but from investigations of biologically active binding-recognition systems. Specificity usually dictates the existence of a rare sequence and, usually only picomole to nanomole quantities are readily available. Mass spectrometry finds its niche in high sensitivity studies. In Section III the reader will find the work of many investigators who apply mass spectrometry to nanomole to picomole amounts of complex carbohydrates. The hard fact is, however, that mass spectrometry alone cannot 36 F. Bollum, " I take a craftsman's view of the practice of science and of the scientist." Personal communication (ca. 1977). 37 Propertius, "Elegies." Book ii (i), p. 43. (51 B.C.) Roughly, "The sailor recants his experiences of great storms, the farmer carries forth about oxen, the soldier talks about wounds and the shepherd discusses sheep."
546
GLYCOCONJUGATES
[29]
yet give a complete structure. It is part of the goal of this introduction and overview to point out the current practical limitations to the analytical methods. Another goal is to suggest strategies and approaches for nanomolar amounts of oligosaccharides that would employ mass spectrometry to lead to complete elucidation of a structure. Many investigators not quoted in this chapter have contributed significantly in this field. It is unrealistic to expect total characterization of intact structures from mass spectra. In the literature, as Biemann notes elsewhere in this volume, 38examples are often taken from known structures which are then "interpreted" according to the ions which happen to occur in the spectra and can be readily explained, often ignoring the unassigned ions. Mass spectra of complete unknowns may not be so simple to interpret. From the empirical descriptions of fragmentation accompanying mass spectra of oligosaccharides, it is readily seen that this is a science in its embryonic stages of development. Despite the extensive early literature, there still seems to be the element of surprise in the origin and mechanism of formation of some fragments. Predictability of fragment ions in spectra according to detailed structure of oligosaccharides is becoming more exact from collision-induced dissociation and tandem mass spectrometry,16'17.2°-29 but it is far from perfect. Example of an Approach to a Biological Carbohydrate The author's particular prejudice lies in the following approach to a total carbohydrate structure (oligosaccharide): "The road to resolution lies by doubt . . . . ,, 39 1. Gain strong evidence for purity. It is virtually impossible to begin with biological extracts and proceed easily to an absolutely pure sample. This derives from the many similar or related structures that are usually present in a biological system. 4° One must be assiduous about this aspect of the analysis. (a) Utilize at least three chromatographic systems, preferably at least one with a derivatized sample, permethylated or peracetylated. (See this series, Volumes 28, 83, 138, 179.) (b) Pay strict attention to composition analysis (methods are described in earlier volumes of this serie s, including Volume s 28, 83, 138, 179) for estimation of size, complexity, and purity. 2. If 50 nmol or more are available, at least obtain a one-dimensional IH NMR spectrum. 3s K. Biemann, this volume [18]. 39F. Quarles, "Emblems." Book iv (No. 2), Epig. 2 (1635). 40C. L. M. Stuits, C. C. Sweeley, and B. A. Macher, this series, volume179, p. 167. Note origins and subtle structural differencesamong 250 glycolipids.
[29]
OVERVIEW AND STRATEGY
547
3. Application of mass spectroscopy: (a) Use 1-20 nmol of the intact compound depending on sensitivity of the instrument, for FAB-MS. (b) Methylate 10-25 nmol (more is desirable). Use 1 or 2 nmol of the methylated derivative for FAB-MS.14 Process the remainder for partially methylated alditol acetates, 4 or alternatively use reductive cleavage4~and process samples by GC/MS, preferably chemical ionization (CI). 5 (c) Utilize Nilsson's technique 42 of periodate oxidation of the intact oligosaccharide, reduction, methylation (or a modification of this method using acetylation, 43 and FAB-MS or direct chemical ionization (DCI). CID may dramatically help this technique, but no report of its application is yet published. One may need 20-50 nmol due to the necessity of carrying the sample through several chemical steps. 4. Determine the remainder of your strategy. For example, you will not know the anomeric configurations or the relative location of linkage positions and anomeric configurations within the oligosaccharide. After following steps 1-3 above, the reader will have knowledge concerning: (a) A reasonable estimation of degree of purity, but not proof. (b) Which sugar(s) belong to nonreducing termini, ff the reader has a free oligosaccharide, it can be reduced with borohydride and the sugar located at the reducing end can be determined. If one methylates this sample, one will also obtain the linkage of the reducing end sugar. Otherwise one must do it the hard way. (c) Sugar composition and stoichiometry, which will give clues regarding purity. (d) Linkage positions and quantity of each sugar type in the molecule (very valuable in estimating purity), but not their relative order in the chain. (e) Whether a branched chain exists in the sample. (f) Whether any sugars have nonsaccharidic substitutions (FAB-MS of the intact saccharide plus FAB-MS of the methylated sampie). (g) Size (molecular weight). The investigator may have some probable knowledge regarding: (a) Location of branch, if any (by FAB-MS and CID cleavages). (b) Anomeric set if NMR has been performed, but not anomeric sequence order. (c) Reducing terminal if oligosaccharide has been reduced. (d) Some information from FAB-MS on the internal order of the sugars, i.e., if it is a heterooligosaccharide, e.g., methylpentose-hexose-N-acetylhexosamine-hexose . . . . The application of mass spectrometry will not provide the investigator with information Concerning the following: (a) Internal sugar order (man-
41 G. R. Gray, this volume [31]. 42 A.-S. Angel and B. Nilsson, this volume [32]. 43 C. G. Hellerqvist, personal communication (1990).
548
GLYCOCONJUGATES
[29]
nose-galactose-glucose) for oligomers where adjacent constituents have the same molecular weight, other than nonreducing terminal(s). (b) If identical sugars with different internal linkages exist, the internal order of the linkages will not be clear. However Nilsson's technique (this volume [32]43 may reveal some information regarding this aspect. (c) Anomeric configurations and their internal order among the constituent sugars. The following steps are necessary to establish the structure: 1. Determine the ring size: As pointed out by Gray in this volume, 41 the 4-substitution of a hexopyranose will give the same partially methylated alditol acetate as the 5-1inked furanose. Notwithstanding that 5-1inked furanoses are either extremely rare or nonexistent in most biological systems, we must prove the structure. Therefore, a few methods exist which can assist with this determination. The one most often used is sequential release of the terminal sugars by glycosylhydrolases (see other volumes in this series: Vols. 28, 83, 138, 179) specific for pyranoses after the terminal residues are identified as to their ring size by methylation linkage analysis. Testing a terminal sialic acid for anomeric linkage is relatively simple. Since biosynthesis has mainly used the tz bond in sialic acids, testing asialidases from one or more sources will usually result in a released sialic acid, revealing the penultimate sugar. When the enzymatically shortened oligosaccharide product is methylated, the ring form of the new nonreducing terminus sugar will be revealed by the Lindberg method. 4 Enzymes specific for this newly revealed terminal sugar can then be used to determine its anomeric specificity and ring size. Another glycosidase will reveal the next sugar, etc. Gray offers an alternative method41: reductive cleavage of the methylated oligosaccharide resulting in preservation of the ring form which can be identified by gas-liquid chromatography/EI-MS. 2. Anomeric set in the absence of NMR data: The most common microscale anomeric determinations are made by a- and fl-glycosylhydrolases when available. Once the sugar composition is known, the enzymes can be tested on the sample followed by chromatography to reveal mobility differences caused by loss of a sugar. As each successive saccharide is removed, a new nonreducing end is available for another exoglycosidase. Occasionally, endoglycosidases are useful. 34Methylation linkage analysis after each successive enzyme release will establish ring size and absolute sequence. Branching or tandem repeated identical residues complicate the problem. Two or more sugars may be removed at once. This raises the question: Were they connected in tandem or on different branches? Obviously, this must be examined by repetitive continuous analysis during enzyme treatment by FAB-MS, preferably with C I D . 16'17 Chemically, chromium trioxide oxidation of the peracetylated oligosac-
[29]
OVERVIEW AND STRATEGY
549
charide in glacial acetic acid has allowed discrimination between O~-D and/3-D bonds. 44 The fl-D-glycosidic linkages has two oxygens in close proximity, the ring and glycosidic oxygen which renders it more susceptible to oxidation by CrO3 than the a bonds. 44 Thus, sugar composition before and after oxidation will assign the survivors as a. Partial survivors suggest mixtures of a and/3 in the sample, either in the same saccharide or in a mixture of sacchafides. CID after chemical ionization was reported to discern among anomers in a complete set of glucose anomeric pairs of each linkage type, using mass-analyzed ion kinetic energy spectra (MIKES), 45 but this discovery has not been developed into a routine system. Solving Difficult Problems
Example I. An extract from a certain tissue may contain a group of 3 to l0 glycosphingolipids with a tetrasaccharide sugar chain, or a glycoprotein may yield as many as 30-60 different N-linked saccharides. Some of these may differ by only one position of linkage, an anomeric configuration or a branch. 4° A fast atom bombardment mass spectrum of such a mixture of several compounds which contain four sugars comprised of galactose, glucosamine, galactosamine, and glucose may be impossible to discern from that of a spectrum of a pure compound. Molecules of the approximate structure Gal-GalNAc-Gal-Glu-ceramide and Gal-GluNAc-Gal-Glu-ceramide may give identical spectra. In fact, they may give identical CID spectra. An example of a mixture consisting of branched and linear compounds includes Gal(fll--+3)
\ /
Gal(fl l--->4)Glu(fl1--*1)ceramide
GalNAc(fl 1---->4) I
Gal(fll--*4)
\
/
Gal(fl 1-*4)Glu(fl 1--*1)ceramide
GalNAc(fl 1--->3) II Gal(cd---)3)
\ /
Gal(fl 1---~4)Gluffl1---~1)ceramide
GalNAc(fl 1---->4) III 44 j. Hoffman, B. Lindberg, and S. Svensson, J. Supramol. Struct. Suppl. 1, 31 (1972). 45 E. G. de Jong, W. Heerma, and D. Dijkstra, Biomed. Mass Spectrom. 7, 127 (1980).
550
GLYCOCONJUGATES
[29]
Gal(fll---)3)GalNAc(fll----~4)Galffll---)4)Glu(fl1--*l)ceramide IV Gal(fl 1---)4)GalNAc(fl l---)4)Gal(fl1---*4)Gluffl1---~1)ceramide V
A mixture like this is difficult to resolve chromatographically and may give mass spectra from which one could draw one of several conclusions. All the members of this family of compounds have the same mass and relative polarity. In fact, they all have identical sugar compositions. The molecular ion may appear to lose a hexose or an N-acetylhexosamine as the nonreducing terminal moiety, or a mixture of the two losses may be seen. The linear compounds IV and V will produce fragments from cleavage of the terminal disaccharide while any of the pure compounds I, II, or III would show no cleavage of a terminal disaccharide, but would show fragments derived from a terminal trisaccharide. Nilsson's method 42 of periodate, permethylation, and FAB could not distinguish among I, II, and Ill, and would have some difficulty with IV because the penultimate GalNAc when linked by either its 3- or 4-position is resistant to periodate. His method is a valid third step, however, after determination of sugar composition, FAB-MS, and methylation linkage analysis. The investigator must be extremely vigilant concerning stoichiometry in analysis, and careful about using a number of different purification methods, both with intact and derivatized samples, utilizing several parallel methods before predicting structure. Dell has suggested one of the most powerful combinations in dealing with unknownsl4: Permethylate the compound; use a part for FAB-MS and a part for methylation linkage analysis. Example 2. Consider a pure compound of the structure: Neu5Ac(~t2---)3)Gal(fll---~4)GluNAc(fll---~4)Gal(fll---~4)Glu-ceramide VI
The strategy used will b e to establish the molecular weight and sugar/lipid composition by FAB-MS, giving a mass which is consistent with the sugar composition listed above and a C18-sphingenine attached to a Czs saturated fatty acid. The FAB spectrum may provide evidence that the sequence of sugar types is as shown above. If the structure were branched, the spectrum may show some differencesJ °,14-17 A methylation linkage analysis 4,5 will establish the composition of the linkage types and a partial structure of VI can be depicted as below: Neu5Ac(ct2--~ ?)Hex ?(fl 1--~4)GluNAc(fl 1--~?)Hex?(fl 1--,4)Hex?-ceramide
where Hex represents hexose. Obviously a large number of possible compounds still remain to be eliminated to find one structure for VI. The
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questions remain because the FAB ionization mass spectrum cannot be used to distinguish among hexoses within the interior of the molecule, in this case o-galactose and o-glucose, although Richter's results look promising. 21,29 Hellerqvist notes 4 that one should take into account the known pathways of biosynthesis for each type of compound. For example, the most common structures in this class of compound have the glucose attached to the ceramide. Thus, a "likely" sequence would be as follows: Neu5Ac(~2---~ ?)Gal(fl l---~4)GluNAc(fl I---~?)Gal(fl 1-~4)Glu-ceramide
However, it should be remembered that if we a r e " completely characterizing" molecules, a "likely" structure is not an absolute one. Nilsson's method 42 helps greatly in this situation. The penultimate galactose will survive periodate oxidation, showing that it is the 3-1inked galactose, the GIcNAc will survive, but this could mean either a 3- or 4-1ink. (Prior hydrazinolysis here would give a free amino group at the 2 position of the GIcNAc. The 4-1inked GIcNAc would become periodate sensitive, rendering the Nilsson technique more useful.) The next galactose will be cleaved between carbons 2 and 3, showing by a specific set of fragments42 that this is the 4-1inked galactose, etc. The question marks remaining point to the fact that the molecule under study (VI) has both a 3- and a 4-1inked o-galactose. Without the Nilsson experiment, 42their order within the chain is not clear. Since the necessary sugar composition analysis shows 2 galactoses, a sequential enzyme degradation would eventually discriminate among the two possible arrangements of galactoses. Removal of the sialic acid followed by methylation would give a 4-1inked and a terminal galactose identifying the original location of the 3-1inked galactose. An exciting possibility is that a set of through-the-ring cleavages with CID and MS/MS might lead to the correct answer. 16.17 Example 3. There are some structures which defy ordinary methods of structural discernment, for example, the lactosamine oligomers shown below. Gal(fl 1---~4)GluNAc(fl 1--~6)
\
Gal(fll--~4)GluNAc(/31---~3)Gal(fll--~4)GluNAc(fll---~3)Gal(fl VII
l--~4)Glu-
Gal(fl l-~4)GluNAc(fl 1---~6)
\
Gal(fl 1--~4)GluNAc(/3 l--~3)Gal(fl 1--~4)GluNAc(fl l--~3)Gal(fl l--~4)GluVIII
Upon sequential methylation analysis and enzyme degradation, a common simple strategy for complete analysis, the products in both cases are identical. Hence, methylation analysis gives the identical result for both, yielding the following: Terminal galactose, 2 mol; 3-substituted galactose,
552
GLYCOCONJUGATES
[29]
1 mol; 3,6-disubstituted galactose, 1 mol; 4-substituted GIcNAc, 3 mol; and 4-substituted glucose, 1 mol. Sequential enzyme degradation with/3galactosidase and/3-hexosaminidase yields the following: galactose, 2 mol and GIcNAc, 2 mol. The fragment IX results from both structures: Gal(fl 1--*4)GluNAc(fll---~3)Gal(fl l---~4)Glu-ceramide iX
R e s u l t . The results of example 3 described above lead to the following conclusions: 1. No combination of methylation linkage analysis and exoglycosylhydrolases can resolve structures VII and VIII. 2. Nilsson's technique cannot resolve structures VII and VIII because the internal residues are all periodate-resistant.42 3. FAB-MS of any of the intact, permethylated or peracetylated compound should give a result which shows that compound VII gives a pentasaccharide B fragment17 while not easily yielding a tetrasaccharide B fragment. Compound VIH would give a tetrasaccharide B fragment and not a pentasaccharide B fragment. Some problems with yield of the fragment could occur because of the propensity for charge retention (inpositive ion FAB-MS) on the GIcNAc in the cleavage reactions such that compound VII could yield a lowabundance pentasaccharide fragment compared with B fragments from cleavages at the GIcNAc glycosidic bonds. A mixture of these two compounds in a sample would be a horrendous analytical enigma. Treating first with/3-galactosidase would give smaller compounds for mass spectrometry which would yield a trisaccharide fragment but not a disaccharide for VII while a disaccharide would be easily obtained from VIII. 46 CID of the fragment ions formed in either would answer the question. Solutions to other difficult carbohydrate analytical problems which may be encountered are given below along with pertinent references. 1. Location of substituents on cyclitols was resolved by Hsieh et al. 47 where an myo-inositol was substituted with both a phosphodiester and a glycosidically linked sugar. The linkages were characterized by a combination of periodate oxidation and analysis of the stereochemically distinct products by gas-liquid chromatography and chemical ionization mass spectrometry. 2. Location of sulfates on heterooligosaccharides was resolved for glycosaminoglycan oligosaccharides by Dell et al. 48 using substitution of 46 K. Watanabe, R. A. Laine, and S.-I. Hakomori, Biochemistry 14, 2725 (1975). 47 T. C.-Y. Hsieh, K. Kaul, R. A. Laine, and R. L. Lester, J. Biol. Chem. 253, 3575 (1978). A. Dell, M. E. Rogers, and J. E. Thomas-Oates, Carbohydr. Res. 179, 7 (1988).
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the sulfates with acetates after permethylation under conditions which retained the sulfates. 3. Hellerqvist e t al. 49 located acyl groups by methyl vinyl ether acetalization5° of free .hydroxyls, an alkali-stable substitution, followed by methyl sulfinyl carbanion and methyl iodide treatment to replace acyl groups with methyl or other alkyl functions. 4. Quantitation of picomole to nanomole amounts of glycosidically bound monosaccharides in complex biological matrices have been attempted by a number of authors. Chemical ionization mass spectrometry was used with gas-liquid chromatography and computer-reconstructed mass chromatograms to analyze N-acetylneuraminic acid directly from erythrocytes by Ashraf e t al. 51 Roboz e t al. 52 and Sugawara e t al. 53 have used similar approaches. Sugiyama e t a l : 4 have also used GC/MS for estimation of free sialic acid. These methods could be adapted for any monosaccharide or partially methylated sugar) The problems of purity, stereochemical differences and identical mass and polarity in mixtures of oligosaccharides make mass spectrometry of this class of compounds most challenging. Still, one can see that enormous progress has been made in technology and ingenuity of approach. We can look forward to more sensitive instruments, novel energy-adding methods for the tandem mass spectrometric methods, and computer fingerprinting of data.
49 C. G. Hellerqvist, B. Lindberg, S. Svennson, and Lindberg, Carbohydr. Res. 8, 43 (1968). 5o A. N. DeBelder and B. Norrman, Carbohydr. Res. 8, 1 (1968). 51 j. Ashraf, D. A. Butterfield, J. Jarnefelt, and R. A. Laine, J. Lipid. Res. 21, 1137 (1980). 52 j. Roboz, R. Suzuki, and J. G. Bekesi, Anal. Biochem. 87, 195 (1978). 53 y . Sugawara, M. Iwamori, J. Portaukalian, and Y. Nagai, Anal. Biochem. 132, 147 (1983). 54 N. Sugiyama, K.-I. Saito, H. Mizu, M. Ito, and Y. Nagai, Anal. Biochem. 170, 140 (1988).
