MASS SPECTROMETRY OF OLIGOSACCHARIDES

Transcription

1 Joseph Zaia* Department of Biochemistry, Boston University School of Medicine, 715 Albany St., R-806, Boston, Massachusetts Received 20 March 2003; received (revised) 6 August 2003; accepted 6 August 2003 I. Introduction II. Characteristics of Tandem Mass Spectra of Carbohydrates A. Ionization of Carbohydrates Electrospray Ionization (ESI) Matrix-Assisted Laser Desorption/Ionization (MALDI) B. Nomenclature for the Fragmentation of Glycoconjugates C. Tandem MS of Native Oligosaccharide Molecular Ions Protonated Ions Deprotonated Ions Alkali and Alkaline Earth Adducted Ions D. Tandem MS of Permethylated and Peracetylated Oligosaccharides E. Tandem MS of Reductively Aminated Carbohydrates F. Discrimination of Monosaccharide Linkages G. Gas-Phase Degradation of Oligosaccharides H. Computer-Based Approaches for Interpretation of Oligosaccharide Product-Ion Mass Spectra I. Internal Residue Loss Rearrangements of Oligosaccharide Ions During CID J. Conclusions III. Analyzers for Mass Spectrometry of Carbohydrates A. Analysis of Permethylated Carbohydrates Using High Temperature GC/MS B. Analysis of Carbohydrates with MALDI-TOF MS C. Analysis of Carbohydrates with MALDI Q-oTOF MS D. Analysis of Carbohydrates with ESI Q-oTOF MS E. Analysis of Carbohydrates with QIT MS F. Analysis of Glycoconjugates with FT MS G. Conclusions IV. Tandem Mass Spectrometry of Glycopeptides A. Ionization of Glycopeptides B. CID of Glycopeptides Selective Identification of Glycopeptides with Tandem MS CID of O-Linked Glycopeptides CID of N-Linked Glycopeptides C. Electron Capture Dissociation of Glycopeptides D. Conclusions V. Mass Spectrometry of Sialylated Glycoconjugates A. Permethylation of Sialylated Oligosaccharides B. MALDI-MS of Sialylated Glycoconjugates Anionic Dopants for Analysis of Sialylated Glycoconjugates Methyl Esterification to Stabilize Sialic Acid Residues Perbenzolylation to Stabilize Sialic Acid Residues High-Pressure MALDI of Sialylated Glycoconjugates Contract grant sponsor: NIH/NCRR; Contract grant number: P41- RR10888; Contract grant sponsor: Glycosciences Research Award, Neose Technologies. *Correspondence to: Joseph Zaia, Department of Biochemistry, Boston University School of Medicine, 715 Albany St., R-806, Boston, MA jzaia@bu.edu Mass Spectrometry Reviews, 2004, 23, # 2004 by Wiley Periodicals, Inc.

2 & ZAIA C. ESI MS of Sialylated Oligosaccharides D. Tandem MS of Sialylated Oligosaccharides E. Conclusions VI. Mass Spectrometry of Sulfated Oligosaccharides A. Derivatization B. Ionization Methods Fast Atom Bombardment MALDI a. MALDI of Sulfated Peptides b. Direct MALDI of Sulfated Oligosaccharides c. Use of Basic Peptides for MALDI of Polysulfated Oligosaccharides d. MALDI Analysis of Protein-Sulfated Oligosaccharide Complexes ESI of Sulfated Oligosaccharides On-Line Separation Systems for Sulfated Carbohydrates C. Tandem MS of Sulfated Oligosaccharides Lessons from CID of Sulfated Peptides Tandem MS of Mono- and Di-Sulfated Oligosaccharides Precursor-Ion and Neutral-Loss Scans for Sulfated Glycoconjugates Determination of Positional Sulfation Isomers in GAG Disaccharides Tandem Mass Spectrometric Quantification of GAG Disaccharides Tandem Mass Spectrometric Analysis of GAG Oligosaccharides a. CS Oligosaccharides b. Heparin/HS Oligosaccharides D. Conclusions VII. Overall Conclusions VIII. Abbreviations References Glycosylation is a common post-translational modification to cell surface and extracellular matrix (ECM) proteins as well as to lipids. As a result, cells carry a dense coat of carbohydrates on their surfaces that mediates a wide variety of cell cell and cell matrix interactions that are crucial to development and function. Because of the historical difficulties with the analysis of complex carbohydrate structures, a detailed understanding of their roles in biology has been slow to develop. Just as mass spectrometry has proven to be the core technology behind proteomics, it stands to play a similar role in the study of the functional implications of carbohydrate expression, known as glycomics. This review summarizes the state of knowledge for the mass spectrometric analysis of oligosaccharides with regard to neutral, sialylated, and sulfated compound classes. Mass spectrometric techniques for the ionization and fragmentation of oligosaccharides are discussed so as to give the reader the background to make informed decisions to solve structure-activity relations in glycomics. # 2004 Wiley Periodicals, Inc., Mass Spec Rev 23: , 2004 Keywords: carbohydrates; oligosaccharides; mass spectrometry; sialylated; sulfated; glycomics I. INTRODUCTION With progress in proteomics comes an increasing interest in the importance of glycosylation. Most cell-surface and secreted proteins are glycosylated a fact that impacts on efforts to understand the biological relevance of specific protein expression and modification patterns. Unlike the core proteins, glycans are expressed as a set of variations on a core structure and are polydisperse in nature. Therefore, glycosylation increases the complexity of protein molecules and causes them to migrate as diffuse bands or spots on SDS PAGE gels to complicate efforts to identify protein expression patterns that correlate with disease states. Glycans serve to modulate protein function and may influence folding, biological lifetime, and recognition of binding partners. For example, surface carbohydrates serve as the interface between the cell and its environment, and define self versus non-self. Many pathogens recognize particular carbohydrates, and structural studies have led to progress in this area. Mass spectrometry is an important tool for the structural analysis of carbohydrates, and offers precise results, analytical versatility, and very high sensitivity. Whereas mass spectrometric analysis options for proteins and peptides are well-defined relative to those for carbohydrates, tandem mass spectrometric product-ion patterns are more complex, and the results depend on the types of derivative and precursor ion used. In most cases, one analyzes protonated forms of underivatized proteins and peptides and collisional-induced dissociation (CID) results 162

3 & in definition of a complete sequence (Biemann & Martin, 1987) or of a partial sequence that is useful in the identification of modified peptide residues (Papayannopoulos, 1995) or in database searching (Mann & Wilm, 1994). With carbohydrates, one has the choice of using peralkyl or reducing terminal derivatives, or of analyzing native structures. One also has the choice of protonated, metalcationized, or deprotonated ions. As a result, the carbohydrate mass spectrometry field is reflected by a divergence of published structural analysis methods in recent years. For protein mass spectrometrists who wish to learn about carbohydrates and glycobiologists who wish to learn mass spectrometry, the plethora of publications that describe seemingly divergent approaches may seem overwhelming. The intent of this review is to summarize the historical and recent developments in carbohydrate mass spectrometry to facilitate informed decision-making on analysis routes. Section II explains common ionization methods used for carbohydrates as well as characteristic fragmentation methods for different carbohydrate compound classes. Section III describes the characteristics of different modern mass analyzers used for carbohydrates. The remaining sections specifically address the analysis of glycopeptides (Section IV), sialylated glycoconjugates (Section V), and sulfated oligosaccharides (Section VI). II. CHARACTERISTICS OF TANDEM MASS SPECTRA OF CARBOHYDRATES A. Ionization of Carbohydrates 1. Electrospray Ionization (ESI) Conventional ESI MS (Meng, Mann, & Fenn, 1988; Fenn et al., 1990) involves the pumping of a solution (a forced flow) into the ion source, and has been observed to produce relatively weak ion signals for native oligosaccharides compared to those for peptides and proteins (Burlingame, Boyd, & Gaskell, 1994; Reinhold, Reinhold, & Costello, 1995). Nano ESI (Wilm & Mann, 1994), on the other hand, produces ion signals that are comparable between the peptide and carbohydrate compound classes (Bahr et al., 1997). It, therefore, appears that the hydrophilicity of oligosaccharides limits the surface activity in ESI droplets and that, with small droplets, their sensitivity is significantly enhanced. The results with nano ESI are consistent with the conclusion that the sensitivity increase observed when oligosaccharides are derivatized, reducing their hydrophilicity, is due to an increase in surface activity rather than in volatility (Karas, Bahr, & Dulcks, 2000). The fact that the ESI of carbohydrates appears to be more effective with the nano scale, has important implications. Interfaces for on-line ESI LC/MS typically produce droplet sizes that are large relative to those produced by spraying from a 1 2 mm orifice nanospray emitter, and thus the spray characteristics are typical of forced flow. An exception appears to be the use of 10 mm fused-silica spray tips with post-column splitting of HPLC effluents (Gangl et al., 2001). It may be possible to design a continuous-flow interface that produces droplet sizes small enough to produce nanospray-like ionization responses for carbohydrates that elute from a chromatography column. 2. Matrix-Assisted Laser Desorption/Ionization (MALDI) The MALDI-TOF ionization efficiency for neutral carbohydrates oligomers has been observed to be constant as the size of the molecule increases, in contrast to that for ESI, where the ionization efficiency decreases with an increasing molecular weight (Harvey, 1993). Although the ionization response drops off with increasing molecular weight for mixtures of large carbohydrate polymers, the use of MALDI for neutral oligosaccharide analysis has advantages over ESI, particularly for applications that involve the profiling of mixtures released from glycoproteins. Quantitation of permethylated carbohydrate mixtures with MALDI has been demonstrated to be reproducible, and the coefficients of variation for those analyses equal those obtained for the same oligosaccharide mixture after derivatization with a chromophore and chromatographic quantitation (Viseux et al., 2001). In addition, information on fucosylation was more detailed with the mass spectrometric approach. The advantages of MALDI in terms of ionization response have to be balanced against the disadvantages of the metastable fragmentation that is caused by the higher internal energies imparted to the ions relative to those resulting from ESI. Although that fragmentation allows the analysis of carbohydrate ions with post-source decay (PSD) on a MALDI-TOF instrument (Viseux, Costello, & Domon, 1999), it complicates MS profiling. Extensive fragmentation of sialic acid residues necessitates either the use of linear mode MALDI-TOF to avoid separating the metastable products (see Section V.D), or of derivatization to stabilize those groups (See Section V.B.2). As a further complication, detection of sialylated glycoconjugates with Fourier transform (FT) MS is limited by the metastable fragmentation in a low-pressure MALDI source and is mitigated through the use of collisional cooling with high pressure (O Connor & Costello, 2001; O Connor, Mirgorodskaya, & Costello, 2002). Atmospheric-pressure sources have been developed for quadrupole ion trap (QIT) instruments (Laiko, Moyer, & Cotter, 2000; Moyer & Cotter, 2002; Moyer et al., 2002), and it is likely that those sources will prove to be useful for the analysis of oligosaccharides. The development of a MALDI TOF/TOF analyzer has been described for the analysis of peptides 163

4 & ZAIA (Medzihradszky et al., 2000; Yergey et al., 2002), and carries the potential benefits of producing high-energy fragmentation. Metastable fragmentation has been a problem with that instrument, and it is likely that its effective use for the analysis of oligosaccharides will require a high-pressure source to limit metastable fragmentation, and thereby allow true high-energy CID. The benefits of high-energy CID of carbohydrates are welldocumented (Domon & Costello, 1988b; Gillece-Castro & Burlingame, 1990; Peter-Katalinic, 1994); see Section II.F for a detailed discussion. B. Nomenclature for the Fragmentation of Glycoconjugates Collisional-induced dissociation (CID) of glycoconjugates results in the observation of ions that correspond to cleavage of the oligosaccharide portion of the molecule. Typically, those ions are produced in greater abundances for the oligosaccharide portion than are those that occur in the aglycon (non-oligosaccharide) portion of glycoconjugates. The nomenclature for oligosaccharide fragmentation used throughout the mass spectrometry field is shown in Figure 1. Fragment ions that contain a non-reducing terminus are labeled with uppercase letters from the beginning of the alphabet (A, B, C), and those that contain the reducing end of the oligosaccharide or the aglycon are labeled with letters from the end of the alphabet (X, Y, Z); subscripts indicate the cleaved ions. The A and X ions are produced by cleavage across the glycosidic ring, and are labeled by assigning each ring bond a number and counting clockwise. Figure 1 shows examples for two cross-ring cleavage ions. Ions produced from cleavage of successive residues are labeled: A m,b m, and C m, with m ¼ 1 for the non-reducing end and X n,y n, and Z n, with n ¼ 1 for the reducing-end residue. Note that Y 0 and Z 0 refer to the fragmentation of the bond to the aglycon. When considering tandem MS of oligosaccharides, there are several options regarding the state of the precursor ion, the choice of which will dramatically influence the product-ion pattern, and thus the structural information, produced. For native oligosaccharides, the options include protonated molecule ions, [M þ nh] nþ, deprotonated molecule ions, [M nh] n, and natriated molecule ions, [M þ Na] þ. Permethylated and peracetylated derivatives are analyzed as either [M þ H] þ or [M þ Na] þ with MALDI, or as multiply charged ions with ESI. Table 1 shows the combination of ionization states and derivatives that have been studied in the literature. Many of the principles of carbohydrate fragmentation with tandem MS were developed with fast atom bombardment (FAB) ionization (also known as liquid secondary ion mass spectrometry), and this review takes those principle into consideration along with later developments. Note that metal-adducted ions, in which charge is carried by protons, will be denoted [M(X) þ H] þ. This nomenclature assumes the adduction of the metal cation with the displacement of a proton, and the positive ion is formed by the addition of a proton. It is understood that the sodium cation is paired with an acidic group. This nomenclature is used here and elsewhere (Costello, Juhasz, & Perreault, 1994; O Connor, Mirgorodskaya, & Costello, 2002; Zaia & Costello, 2003) because it simplifies the notation of precursor and product ions, and facilitates the labeling in tandem mass spectra. Note that cationized ions, in which charge is carried by the metal, are given as [M þ mx] mnþ, where m ¼ the number of metal ions and n ¼ the cationic charge. Tandem MS of carbohydrates was first studied with FAB ionization, a technique that imposed severe constraints on the analysis. Neutral (containing no amino groups) and basic (containing hexosamine residues) carbohydrates ionize relatively poorly with FAB, and the tandem MS analysis was practically limited to molecules of <1000 Da. Acidic oligosaccharides (those containing Neu5Ac or sulfate) were observed to produce relatively strong ions with negative FAB (Egge & Peter-Katalinc, 1987). The ion signals were observed to be much stronger when the oligosaccharides or glycoconjugates were permethylated or peracetylated. The FAB ionization process was also energetic enough to produce glycosidic-bond and cross-ring cleavages a phenomenon that was used to obtain structural information on molecules that had been purified to homogeneity (Dell et al., 1983a,b; Egge, Dell, & Von Nicolai, 1983; Carr & Reinhold, 1984). It was also observed that neutral gas collisions enhanced the yield of fragment ions generated from oligosaccharides (Carr et al., 1985). The development of tandem mass analyzers for TABLE 1. Ionization options for tandem MS of oligosaccharides FIGURE 1. Nomenclature for glycoconjugate product ions generated by tandem MS (Modified from Domon & Costello, 1988b). 164

5 & biological macromolecules allowed the selection of individual precursor ions and subsequent fragmentation, thus eliminating the need to purify oligosaccharides to homogeneity. Using FAB tandem MS, it became generally accepted to analyze sodium adducts of permethylated oligosaccharides as [M þ Na] þ ions as will be discussed in detail below. The development of ESI greatly expanded the capabilities of the tandem MS of oligosaccharides by improving, relative to FAB, the strength of the ion signals produced from a given quantity of oligosaccharide. Although the permethylated oligosaccharides were observed to produce the strongest signals, the direct analysis of native oligosaccharides was possible for larger precursor ions than was possible with FAB. That increase in sensitivity was such that researchers had the choice to ionize native or permethylated oligosaccharides or glycoconjugates as protonated, deprotonated, or alkali-adducted ions. Because the information content of product-ion mass spectra depends to a large extent on the state of the precursor ion, it is important to examine the available information in this area. The following sections summarize the fragmentation behavior of oligosaccharides and oligosaccharide portions of glycoconjugates with regard to the state of the precursor ion so as to allow the reader to choose the appropriate analysis options. Recent work in this area is summarized, with regard to overall trends for carbohydrate fragmentation. Product-ion mass spectra of glycoconjugates are considerably more complicated than are those of peptides because of their branching structure. For such branching structures, fragmentation occurs from the non-reducing end of each antenna and from the reducing end to give rise to a higher complexity. The input of energy into the molecule by collision is most likely to break single bond glycosidic linkages. Using low energy CID, fragmentation of glycosidic linkages is most likely; fragmentation across the sugar rings is less so because two covalent bonds must be cleaved. It does occur, however, and provides important information on the location of substituents on branching monosaccharide residues. Research has shown that CID product-ion mass spectra provide information on the stereochemistry of individual sugar residues (Mueller et al., 1988), the linkage position (Laine et al., 1988), and branching structure (Carr et al., 1985; Domon & Costello, 1988a). Oligosaccharides that contain the same monosaccharides linked with a different branching structure often show distinct product-ion patterns because the steric environments differ between such isomers and result in different bond energies and ion abundances in product-ion mass spectra (Laine et al., 1988). Today, it is not possible to determine all of the linkages and branching patterns for a complex branched oligosaccharide. However, much work has been conducted in the past 15 years to improve the degree to which the product-ion patterns generated from those molecules may be directly interpreted, as opposed to their being used as mass fingerprints, whose pattern reflects but does not predict the structure. C. Tandem MS of Native Oligosaccharide Molecular Ions 1. Protonated Ions The CID fragmentation patterns differ according to the oligosaccharide sequence, size, and class of subunits, as discussed below, and are used to differentiate subtle structural differences. CID fragmentation of [M þ H] þ ions generated from asialo glycoconjugates results in the observation of abundant B m and Y n ions; those patterns are useful in assigning the oligosaccharides sequence (Domon & Costello, 1988b). Cleavage to the reducing side of HexNAc residues is favored, resulting in abundant fragment ions (Egge, Dell, & Von Nicolai, 1983). Therefore, such fragmentation is useful to define the presence of lactosamine (Gal-GlcNAc) disaccharides in the antennae of N-linked oligosaccharides. CID fragmentation of neutral glycoconjugates (gangliosides) results in the observation of abundant B m and Y n ions, indicating that charge is not localized in those ions (Domon & Costello, 1988b). The abundances of ions produced from cross-ring cleavages is comparatively low for [M þ H] þ ions generated from that compound class. Those results are similar to the metastable fragmentation observed for those molecules in FAB MS (Egge & Peter-Katalinc, 1987; Gunnarson, 1987). When the protonated ions generated from small oligosaccharides are subjected to CID, a greater diversity of information is produced than that obtained from large oligosaccharides (Dell, 1990; Gillece-Castro & Burlingame, 1990). Specifically, glycosidic bond cleavages are more likely to predominate in product-ion mass spectra of large ions, with the result that less detailed information regarding the branching structure is obtained. Comparatively more subtle information, with regard to cross-ring cleavage ions, may thus be obtained for small ions. Those observations have led to efforts to increase the information content of product-ion mass spectra. Conditions during ESI MS of native oligosaccharides can be manipulated to favor the production of protonated, natriated, or ammoniated precursor ions (Duffin et al., 1992). Natriated oligosaccharides are produced in the presence of a low concentration of sodium acetate. Ammoniated adducts formed in the presence of ammonium acetate readily decay to protonated ions due to the energy required to desolvate ions during the electrospray process. Sialylated oligosaccharides were observed to produce abundant [M H] ions in the negative-ion mode with ESI MS. The yield of glycosidic bond product ions in high-energy CID 165

6 & ZAIA was observed to be higher for protonated relative to natriated native oligosaccharide ions (Orlando, Bush, & Fenselau, 1990; Duffin et al., 1992). Cleavages to the reducing side of HexNAc residues are observed in abundance, particularly to those residues located on complex N-linked antennae. High-mannose oligosaccharides produce a series of product-ions from successive losses of hexose residues, resulting in a generally more uniform product ion pattern than observed for complex N- linked sugars that contain HexNAc residues near the nonreducing termini. Thus, the pattern of CID product ions is useful to define the class of N-linked oligosaccharide (highmannose, hybrid, or complex). 2. Deprotonated Ions Neutral oligosaccharides have been observed to ionize relatively poorly relative to their basic (amino sugarcontaining) and acidic counterparts. Sialylated oligosaccharides are amenable to analysis as [M H] ions, and are observed to produce product ions as abundant as those from metastable fragmentation with FAB ionization (Derappe et al., 1986; Egge & Peter-Katalinc, 1987). A series of molecules with Neu5Ac at the non-reducing terminus fragment to produce ions that almost exclusively contain the acidic group. This is contrasted with the fragmentation of an asialo ganglioside in which both reducing and non-reducing fragmentation results (Domon & Costello, 1988b). Fragmentation to the reducing side of HexNAc is facile in the CID of [M H] ions. The importance of mobile protons in analyzing the fragmentation pattern becomes evident when comparing studies of deprotonated polysaccharides with their protonated forms. Interestingly, the high-energy CID production mass spectrum of the [M H] ion generated from maltoheptaose (1,4-linked glucose heptamer) displays only 2,4 A m ions (Gillece-Castro & Burlingame, 1990). By contrast, the [M H] ion produced from the chitobiose core oligosaccharide, a branched (Man) 2 -Man-GlcNAc- GlcNAc structure, displays abundant cross-ring cleavages with limited glycosidic bond fragmentation. The linear maltoheptaose spectrum displays a smooth decrease in ion abundances as the m/z values decrease, whereas the pattern generated from the chitobiose core is defined by a very abundant 2,4 A 3 ion. The discontinuous product-ion pattern of the chitobiose indicates branching. Protonated ions generated from the same molecules produce very abundant glycosidic bond cleavages with the very low abundance of cross-ring cleavages. The profusion of glycosidic bond cleavages of native oligosaccharides in CID product-ion mass spectra seems to be related to the presence of free hydroxyl groups. Hydroxylic hydrogen migration has been implicated in glycosidic-bond cleavages (Hofmeister, Zhou, & Leary, 1991), and the abundance of such ions appear to limit the abundance of cross-ring cleavage ions. In accordance, certain cross-ring cleavage ions, observed in the production mass spectra of permethylated oligosaccharides, have been observed to be absent from spectra acquired on native oligosaccharides (Harvey, Bateman, & Green, 1997). In protonated or alkali-cationized oligosaccharides, the presence of HexNAc residues places a charge on the amide nitrogen. The proximity of that charge to the reducing-side glycosidic oxygen evidently predisposes that bond to scission. Although a similar pattern is observed for deprotonated oligosaccharides, it is not observed for chondroitin sulfate glycosaminoglycans which consist of [UroA(1 3)GalNAcSulfate(1 4)] n, in the negative ESI mode (Zaia, McClellan, & Costello, 2001; McClellan et al., 2002). The CID production mass spectrum of a triply charged CS hexamer (n ¼ 3) results in abundant glycosidic bond cleavages to the reducing side of uronic acid residues but not to the reducing side of GalNAc residues. This difference is evidence that the glycosidic bond to the reducing side of GalNAc is significantly less labile than is the bond to the reducing side of UroA residues. This result is in marked contrast to the observations made for deprotonated oligosaccharides with FAB MS (Domon & Costello, 1988b). For further discussion on this issue, see Section VI.C.6.a. 3. Alkali and Alkaline Earth Adducted Ions The fragmentation of natriated native oligosaccharides by high-energy CID results in different product-ion patterns than are obtained from protonated ions (Orlando, Bush, & Fenselau, 1990). Specifically, the production of cross-ring cleavages is enhanced relative to the levels observed in the product-ion mass spectra of protonated molecular ions. Furthermore, the abundance of ions produced from cross-ring cleavages were reduced as the collision energy was lowered from 8 to 2 kev; that reduction indicates that the use of high-energy CID increases the information content of product-ion spectra. The Low-energy CID spectra of monolithiated disaccharides show abundant reducing end cleavages, but little differentiation between different linkages. Dilithiated disaccharides produce product-ion patterns that readily differentiate linkage isomers (Zhou, Ogden, & Leary, 1990). The dilithiated ions have lithium substitution for a hydroxyl hydrogen, and that substitution is believed to initiate the ring-cleavage rearrangements. Using [M(Li) þ Li] þ ions, it was possible to assign the linkages of Glc(1 6)Glc(1 6)Glc(1 6)Glc versus the (1 4) linked isomer. PSD allows the analysis of product-ions that result from low-energy processes. PSD of natriated ions produced from native oligosaccharides resulted in an abundant series of glycosidic-bond and cross-ring cleavages. 166

7 & Cleavages adjacent to HexNAc residues were facile, as observed for other dissociation techniques (Spengler et al., 1994). PSD analysis of natriated and protonated forms of Gal(b1-3)-GlcNAc(b1,3)-Gal(b1-3)-Glc-Bz (benzyl amino derivative) is instructive (Lemoine, Chirat, & Domon, 1996). The natriated form produces abundant B m and Y n ions, indicating that charge is located without preference to either end of the molecule. The protonated form produces primarily Y n ions, indicating that the charge resides on the basic benzylamino group on the reducing terminus. The B 2 ion is also observed from facile cleavage of the GlcNAc residue. PSD of a series of protonated N- and O-linked benzylamino structures was also shown to result in predominantly Y n ions. MALDI FTMS studies on alkali cationized native oligosaccharides have shown that the yield of fragmentation correlates with the degree of branching (Cancilla et al., 1996). Fragmentation yields were highest for oligomers with the least branching, and were inversely related to cation size, following the order H þ >Li þ >Na þ >K þ > Rb þ >Cs þ. Mechanism offragmentation of protonated ions is likely to be charge-induced, whereas that for cesiated ions is likely to be charge-remote. Charge-remote fragmentation requires more energy than charge-induced fragmentation, and the degree to which this occurs increases with increasing cation size. The effects of protons versus alkali metal cations may be rationalized based on differences in the coordination of the glycosidic oxygen. As shown in Figure 2, protonation is likely to be localized to the glycosidic oxygen the most basic in the structure (Cancilla et al., 1996). Metal ions, on the other hand, can undergo coordination with several atoms simultaneously, resulting in less destabilization of the glycosidic bond. This result is consistent with a higher barrier to glycosidic-bond fragmentation for the alkali cationized ions. In addition to the glycosidic oxygen atom, acidic residues coordinate alkali metal cations, and this factor has been investigated as a means for stabilization and linkage determination (Penn, Cancilla, & Lebrilla, 2000). CID product-ion mass spectra of sialylated milk oligosaccharides were acquired on [M(X) þ X] þ ions, where X ¼ Li, Na, K, or Cs, and showed that sialic acid residues are able to bind two cesium cations but only one lithium or sodium cation. It appears that small cations, with a high charge density, repel each other, making binding of two cations energetically unfavorable. Alkali-metal cationization and subsequent positiveion CID MS results in three possible fragmentation pathways, as shown in Figure 3 (Cancilla et al., 1999). The complex may dissociate to lose the metal cation (path A), leaving the oligosaccharides as a neutral, and producing no detailed information. The complex may undergo glycosidic bond cleavage (path B) with cation retention by one of the fragments. The complex may also undergo cross-ring cleavage (path C). The likelihood of dissociation of the cation increases with size, and is greatest for Cs þ and least for Li þ and Na þ. Activation energies for glycosidic-bond cleavage have been determined to be lowest for Li þ - coordinated oligosaccharides. As a result, use of this cation maximizes glycosidic bond fragmentation. The low energy barrier results in the observation of abundant fragment ions by MALDI MS, particularly for FT analyzers for which the ion lifetime is long. Glycosidic-bond cleavages are believed to be charge-induced, and Li þ appears to mimic protons by having high charge density and associating with glycosidic oxygen atoms. The use of Cs þ is best for producing MALDI ions stable enough to be detected in instruments, such as the FTMS, with long ion lifetimes. That cation is large and interacts with several sites on the oligosaccharide to minimize the destabilization of the glycosidic bond (Tseng et al., 1997). Activation energy barriers have been studied with semiquantative methods, and have been found to be independent of alkali metal ions. Those cleavages appear to be charge-remote, occurring some distance from the charged site. Glycosidic-bond FIGURE 2. Fragmentation of (a) protonated and (b) alkali-cationized glycosidic bonds (Modified from Cancilla et al., 1996). 167

8 & ZAIA FIGURE 3. Three possible fragmentation pathways for metal-cationized oligosaccharides. Reprinted with permission of Cancilla et al. (1999). Copyright 1999 American Chemical Society. cleavages appear to be favored under conditions where a cation is associated with a glycosidic oxygen atom. Crossring cleavages occur under conditions where the cation binds simultaneously to several oxygen atoms and the degree of destabilization of glycosidic linkages is minimized. Analysis of protonated and natriated N-linked oligosaccharides by ESI Q-oTOF MS resulted in the conclusion that the latter produces the more-informative product-ion profiles (Harvey, 2000a). Cross-ring cleavages were observed to the reducing terminal GlcNAc residue and, at low abundances, to antenna residues. The abundance of crossring cleavage ions diminished with increasing number of HexNAc residues in the antennae; that result is consistent with the idea that cross-ring cleavages are most abundant in the absence of labile glycosidic bonds. During CID of an oligosaccharide molecular ion, the transfer of energy to rotational and vibrational modes competes with glycosidic-bond cleavage (Mendonca et al., 2003). The degree to which peralkylated oligosaccharides undergo glycosidic bond cleavage increases with the size of the alkyl group; i.e., methyl<ethyl<propyl<butyl< pentyl. The larger alkyl groups are less free to rotate about the glycosidic bonds, and therefore dissociate at a higher rate to relieve steric and torsional strain. Certain rotational states are inaccessible for large alkyl groups, so that transition states that lead to dissociation are ordered and give rise to reduced energies of activation. The smaller derivatives will have a less-ordered transition state and thus higher entropy of activation. The anomeric configuration of (1 4) linked disaccharides can also be distinguished by the CID of peralkylated protonated ions (Mendonca et al., 2003). Product ion-to-parent ion ratios are higher for the b-isomer (cellobiose) than the a-isomer (maltose) for all alkyl groups. The ratios reflect the differences in crowding between a- and b-glycosidic linkages. The degree of discrimination is highest for the largest alkyl group tested (pentyl), reflecting the greater degree of conformational restriction. The anomeric differentiation in (1 6)-linked peralkyl derivatives is not observed due to the high conformational flexibility of this linkage. Unambiguous determination of linkages of increasingly large oligosaccharides requires the coordination with relatively more equivalents of metal ions, as shown with ESI (Fura & Leary, 1993). In addition to the amount, the choice of metal is important for large oligosaccharides. Alkaline earth elements (Mg 2þ and Ca 2þ ) should, when coordinated with oligosaccharides, produce similar product-ion profiles as dilithiated compounds. Lithium has a high charge-to-radius ratio and is similar to Mg 2þ in that both ions have a small radius and form stable compounds with small anions. Calcium ions, with a larger radius, are expected to coordinate more efficiently with branched oligosaccharides. Note that [M þ Na] þ and [M þ K] þ ions are isobaric with [M þ Mg H] þ and [M þ Ca H] þ ions, respectively. Thus, the use of isotopically pure 26 Mg and 44 Ca may be necessary to establish that an observed adduct is due to the divalent cation rather than to sodium or potassium. As the concentration of oligosaccharide decreases, the optimal metal-to-oligosaccharide ratio increases. For example, at 1.5 nmol/ml, the optimum ratio is 1:1, but at 12 pmol/ml, the ratio is 10:1 (Fura & Leary, 1993). Results of the CID of coordinated trisaccharides were much clearer for Ca 2þ than for Mg 2þ. Here, Ca 2þ formed only a few well-defined product ions, whereas the latter produced complex patterns due to the presence of competing disaccharide pathways. The radius of Mg 2þ (0.65 Å) is much smaller than that of Ca 2þ (0.99 Å) and is thus expected to coordinate many more sites on the trisaccharide molecule. Binding of Ca 2þ is more selective in its sites of coordination, and thus produces simpler product-ion patterns. Coordination with Mg 2þ results in more abundant cross-ring cleavages than do two equivalents of Li þ due to the increased ability of the divalent ion to polarize and bind tightly. Tighter binding leads to more cross-ring cleavages. With larger oligosaccharides, Li þ induces product-ion patterns that are similar to those by H þ, giving rise to only glycosidic bond cleavages (Fura & Leary, 1993). The abundance of natriated ions is maximized at high cone voltages (Harvey, 2000a) conditions that also result in in-source fragmentation. Because of this factor, the precursor-ion pattern is not necessarily reflective of the glycan composition. With divalent metals (Ca 2þ,Mg 2þ, Co 2þ,Cu 2þ,Mn 2þ ), the dominant species formed from chloride salts were [M þ X] 2þ (Harvey, 2001). The ability of divalent metal ions to ionize neutral oligosaccharides follows the order Ca 2þ > Mg 2þ > Mn 2þ > Co 2þ > Cu 2þ. The metal-adducted oligosaccharides resulted in singly and doubly cationized product ions with abundances 168

9 & highest for the Ca 2þ ions. Because 1þ and 2þ product ions are present simultaneously, the CID profiles are relatively complex. The type of fragment ions observed for N-linked oligosaccharides are similar to those observed with natriated ions in that the only cross-ring cleavage ions observed in abundance were 0,2 A and 2,4 A of the reducingterminus GlcNAc residue. The primary advantage of divalent cations, the high relative abundance of the adducted ion, particularly for Ca 2þ, is offset by the increased complexity of the CID spectra. At the time of this writing, Co 2þ (Sible, Brimmer, & Leary, 1997) and Ca 2þ (Harvey, 2001) are the most promising divalent metals for CID of native oligosaccharides. D. Tandem MS of Permethylated and Peracetylated Oligosaccharides The conversion of glycans to hydrophobic derivatives enhances their signal strengths regardless of the ionization technique used. The two most common derivatization procedures involve peracetylation (Bourne et al., 1949) and permethylation (Ciucanu & Kerek, 1984); both can be carried out in high yield (Dell, 1990). Permethylation has come to be the preferred method because it results in a smaller mass increase and a greater volatility (McNeil et al., 1982; Dell et al., 1983a,b). Peracetylated derivatives may be detected in the positive mode as [M þ Na] þ ions; CID fragmentation results in preferential cleavages to the reducing side of HexNAc residues (Dell et al., 1983). As with permethylated derivatives, [M þ H] þ ions from peracetylated oligosaccharides fragment to produce little multiple-bond fragmentation (Domon, Müller, & Richter, 1990). This result is an important advantage because the m/z values of such ions are, in many cases, not distinguishable from those of primary fragment ions generated from underivatized oligosaccharides. Therefore, internal fragment ions produce unique mass values in permethylated and peracetylated oligosaccharide product-ion mass spectra. As a consequence, linear and branched oligosaccharides are easily distinguished for peracetylated [M þ H] þ ions despite the lack of crossring cleavage ions (Domon, Müller, & Richter, 1990). Permethylation is often preferred over peracetylation; for example, for the oligosaccharides with free hexosamine amino groups that characterize the glycan core of glycoinositol phospholipid anchor. Those residues will be rendered indistinguishable from those that are acetylated if they are peracetylated. Permethylation produces a quaternary ammonium cation from the free amine groups that is differentiated by mass from the N-acetylhexosamine residues, and also gives rise to unique CID fragmentation (Baldwin et al., 1990). Permethylated oligosaccharides are most often ionized as [M þ Na] þ ions and produce reducing and non-reducing terminal product ions with approximately equal abundances and preferential fragmentation to the reducing side of HexNAc residues (Egge, Dell, & Von Nicolai, 1983). Fragmentation of the substituents linked to the 3-position of HexNAc residues is also preferred (Egge & Peter- Katalinc, 1987). In general, ions that correspond to Neu5Ac oxonium ions are abundant in fragmentation spectra of sialylated oligosaccharides and glycoconjugates, in some cases complicating the task of locating the position of substitution because ions with glycosidic bonds to those residues are in low abundance. Fragmentation of [M þ H] þ generated from permethylated oligosaccharides also produces useful CID production mass spectra. Cleavage to the reducing side of HexNAc is facile, and subsequent stages of MS are quite useful for differentiating isomers. The protonated molecule ions produce exclusively glycosidic-bond cleavage with lowenergy CID on a triple quadrupole instrument (Viseux, de Hoffmann, & Domon, 1997). Although multiple bond fragmentation is observed, permethylation allows the ions to be unambiguously identified on the basis of the exposed free hydroxyl groups. HexNAc residues substituted at position- 3 undergo a specific elimination that identifies that type of interglycosidic linkage. Tandem mass spectrometric analysis with MS n of permethylated N-linked oligosaccharides has been facilitated by removal of labile groups. The most labile groups are Neu5Ac and HexNAc residues for typical oligosaccharides, and subsequent stages of MS proved information on the non-reducing terminus and core structure that is not obtained in the MS 2 profile (Weiskopf, Vouros, & Harvey, 1997, 1998; Reinhold & Sheeley, 1998; Sheeley & Reinhold, 1998; Viseux, de Hoffmann, & Domon, 1998). Exposure of core structures produces a fragment-ion pattern upon subsequent CID that appears to be independent of the history of formation of the ion. Figure 4 compares product ions of m/z , (GlcNAc) 2 (Man) 3, generated by MS 4 of a sialylated biantennary structure and by MS 3 of asialo biantennary structure NA2 (the specific fragmentation routes are shown in the figure). The structure of the non-reducing antennae may also be elucidated by MS n, producing linkage information based on the presence of cross-ring cleavages, and/or comparison to reference mass spectra (Reinhold & Sheeley, 1998; Sheeley & Reinhold, 1998; Weiskopf, Vouros, & Harvey, 1998). The protonated ions fragment at energies < natriated ions, and the product-ion patterns contain different features. Permethylated oligosaccharides may be desalted with reversed phase chromatography, resulting in their ionization as protonated species (Laine et al., 1988, 1991; Viseux, de Hoffmann, & Domon, 1997, 1998). For example, Figure 5 shows (a) the ESI mass spectrum of milk oligosaccharide lacto-n-tetraose [Gal(b1-3)GlcNAc(b1-3)Gal(b1-4)Glc), LNT], (b) MS 2 of the protonated ion, 169

10 & ZAIA FIGURE 4. MS n fingerprinting of N-linked oligosaccharide core for facile determination of sugar branching and substitution: (a)ms 4 of the glycan core of the disialo, biantennary human transferrin glycan, m/z ; (b) MS 3 of the glycans core of the asialo, galactosylated biantennary oligosaccharide (NA2) from human fibrinogen, m/z Reprinted with permission from Weiskopf, Vouros, & Harvey (1998). Copyright 1998 American Chemical Society. and (c) MS 2 of the natriated ion. The protonated ion fragments to produce fragments that contain the nonreducing terminus. The natriated ion, on the other hand, fragments to produce abundant B m and Y n ions. The protonated ions undergo eliminative losses of substituents to the 3-position of GlcNAc to produce an E m ion. Those ions result from facile fragmentation of corresponding B m ions, as shown in Figure 6, and are useful to identify the mass of the substituent on the 3-position. If the 3-position is unsubstituted, then the loss of a methoxy group is observed. If a monosaccharide residue is located on the 3-position, then the E m ion will reflect that loss. The E m ions were not observed for natriated ions. Fragmentation of protonated permethylated oligosaccharides, therefore, appears to have significant advantages over the natriated variants, by virtue of the specific losses that indicate the pattern of substitution of HexNAc residues. Unfortunately, those advantages are offset by other problems, such as internalresidue rearrangements (see Section II.I). An LC/MS approach has been developed for the online analysis of permethylated oligosaccharides with an ion trap (Delaney & Vouros, 2001). The released oligosaccharides were reductively aminated with 2-aminobenzamide to facilitate UV detection and permethylated. Ions were subjected to data-dependent MS 2 as they eluted from a reversed phase HPLC column as sodium adducts. This analysis entails the software-controlled detection of ions followed by isolation and CID of those ions that meet abundance or other exclusion criteria. The most abundant product ion was automatically selected for MS 3. The HPLC-separation step carries the advantage that isomeric structures are likely to be separated to some degree. As in other QIT MS n analyses, the abundance of cross-ring cleavages are quite low. The data are, therefore, most useful as mass fingerprints, and the HPLC separation should, therefore, facilitate the building of a library of reference structures in a manner similar to that published (Viseux, de Hoffmann, & Domon, 1998; Tseng, Hedrick, & Lebrilla, 1999). E. Tandem MS of Reductively Aminated Carbohydrates The reducing-terminus aldehyde group of oligosaccharides is easily reacted with alkylamines. As shown in Figure 7, the amine forms a Schiff base with the aldehyde that cyclizes to form a glycosylamine. The Schiff base is usually reduced to form a secondary amine because this amine is more stable than a glycosylamine. Such a reductive amination of glycans provides several benefits for MS analysis. The derivative usually contains a chromophore to enhance its chromatographic detectability. Derivatives that 170

11 & FIGURE 6. Fragmentation of permethylated N-acetylhexosamine-containing oligosaccharide B ions, glycosylated in the 3-position (a) and in the 4-position (b). Reprinted with permission from Viseux, de Hoffmann, & Domon (1997). Copyright 1997 American Chemical Society. FIGURE 5. ESMS spectrum of permethylated LNT (a), and ESMS/MS spectra of protonated (b), and natriated (c) molecular species measured at collision offset voltages of 15 and 40 V, respectively. Reprinted with permission from Viseux, de Hoffmann, & Domon (1997). Copyright 1997 American Chemical Society. increase the hydrophobicity of the oligosaccharide will typically increase signal intensity obtained from any ionization technique (Wang et al., 1984; Carr et al., 1985; Gillece-Castro & Burlingame, 1990; Poulter & Burlingame, 1990; Harvey, 2000b). A number of amine groups have been used for reductive amination in conjunction with ESI CID MS, and their structures are shown in Figure 8 (Harvey, 2000b). The choice of the amine influences the ionization efficiency, and the effects are different with MALDI versus ESI, as shown in Figure 9. Although the more-hydrophobic derivatives tend to produce better ionization responses for both ionization modes, the 2-aminoacridone derivative produces a remarkable difference in response between the two ionization techniques. That derivative is widely used in fluorophore-assisted carbohydrate electrophoresis, and has been used in mass spectrometric studies (Okafo et al., 1997). It should be noted that the ESI measurements in Figure 9 were made at flow rates of nl/min, where the droplet size is closer to high flow rate rather than nano flow. It is, therefore, possible that the less hydrophobic derivatives will produce stronger ion signals with nanospray, just as was observed with underivatized oligosaccharides (Bahr et al., 1997; Karas, Bahr, & Dulcks, 2000), resulting from the increased surface activity from smaller droplet size. The nature of the reductive amination derivative was found to have comparatively little effect on the CID profiles of natriated ions, indicating that the cation associates with the monosaccharide residues rather than with the derivative (Harvey, 2000b). The profiles were also similar to those of natriated native oligosaccharides. Crossring cleavages were present in low relative abundances, requiring extended summation times to achieve an adequate signal-to-noise ratio for proper identification. F. Discrimination of Monosaccharide Linkages High-energy CID of cationized permethylated oligosaccharides furnishes information that identifies (2 3) versus (2 6) linked Neu5Ac residues, as well as (1 3) versus (1 4) linked hexose on a HexNAc residue (Lemoine et al., 1991). Discrimination between Neu5Ac linkages is based on the different affinities for sodium cations that gives rise 171