Metal ion adducts in the structural analysis of ginsenosides by electrospray ionization with multi-stage mass spectrometry

The effect of metal (Li+, Na+, K+, Ag+) cationization on collision-induced dissociation of ginsenosides was investigated by electrospray ionization mass spectrometry combined with multi-stage mass spectrometry (ESI-MS(n)). The fragments of sodiated and lithiated molecules give valuable structural information regarding the nature of the aglycone and the sequence and linkage information of sugar moieties. However, the number and relative abundances of fragment ions from lithiated ginsenosides are significantly greater than for the sodiated species. The K+ adducts undergo glycosidic cleavages and very limited cross-ring reactions. The silver ion adducts fragment mainly through glycosidic cleavages.     

Electrospray mass spectrometry and fragmentation of N-linked carbohydrates derivatized at the reducing terminus

Derivatives were prepared from N-linked glycans by reductive amination from 2-aminobenzamide, 2-aminopyridine, 3-aminoquinoline, 2-aminoacridone, 4-amino-N-(2-diethylaminoethyl)benzamide, and the methyl, ethyl, and butyl esters of 4-aminobenzoic acid. Their electrospray and collision-induced dissociation (CID) fragmentation spectra were examined with a Q-TOF mass spectrometer. The strongest signals were obtained from the [M + Na]+ ions for all derivatives except sugars derivatized with 4-amino-N-(2-diethylaminoethyl)benzamide which gave very strong doubly charged [M + H + Na]2+ ions. The strongest [M + Na]+ ion signals were obtained from the butyl ester of 4-aminobenzoic acid and the weakest from 2-aminopyridine. The most informative spectra were recorded from the [M + Li]+ or [M + Na]+ ions. These spectra were dominated by ions produced by sequence-revealing glycosidic cleavages and "internal" fragments. Linkage-revealing cross-ring cleavage ions were reasonably abundant, particularly from high-mannose glycans. Although the nature of the derivative was found to have little effect upon the fragmentation pattern, 3-aminoquinoline derivatives gave marginally more abundant cross-ring fragments than the other derivatives. [M + H]+ ions formed only glycosidic fragments with few, if any, cross-ring cleavage ions. Doubly charged molecular ions gave less informative spectra; singly charged fragments were weak, and molecular ions containing hydrogen ([M + 2H]2+ and [M + H + Na]2+) fragmented as the [M + H]+ singly charged ions with no significant cross-ring cleavages.     

Influence of the labeling group on ionization and fragmentation of carbohydrates in mass spectrometry

The ionization and fragmentation behaviors of carbohydrate derivatives prepared by reaction with 2-aminobenzamide (AB), 1-phenyl-3-methyl-5-pyrazolone (PMP), and phenylhydrazine (PHN) were compared under identical mass spectrometric conditions. It has been shown that the intensities of signals in MS spectra depend on the kind of saccharides investigated and reducing end labels used. PMP sialyllactose, when ionized by ESI/MALDI, produced a mixture of [M + H]+, [M + Na]+, [M - H + 2Na]+ ions in the positive mode and [M - H]-, [M + Na - 2H]- ions in the negative mode. The AB and PHN derivatives formed abundant [M + H]+ and [M - H]- ions in ESI, and by matrix-assisted laser desorption/ionization (MALDI) produced abundant [M + Na]+ ions. PMP- and reduced AB-sialyllactose produced only Y-type fragment ions under both MS/MS sources. In the electrospray ionization (ESI)-MS/MS spectrum of PHN-sialyllactose, abundant ions corresponded to B, Z cleavages and in its MALDI-MS/MS spectrum, the abundant ions were consistent with Y glycosidic cleavages with the concurrence of B, C, and cross-ring fragment ions. In the MALDI-MS spectra of oligosaccharides acquired immediately after derivatization, it was possible to detect only PHN derivatives. After purification, spectra of all three types of derivatives showed high signal-to-noise ratios with the most abundant ions observed for AB reduced saccharides. [M + Na]+ ions were the dominant products and their fragmentation patterns were influenced by the type of the labeling and the kind of oligosaccharide considered. In the MALDI-PSD and -MS/MS spectra of AB-derivatized glycans, higher m/z fragment ions corresponded to B and Y cleavages and the loss of bisecting GlcNAc appeared as a weak signal or was not detected at all. Fragmentation patterns observed in the spectra of hybrid/complex PHN and PMP glycans were more comparable-higher m/z fragments corresponded to B and C glycosidic cleavages. For PHN glycans, the abundance of ions resulting from the loss of bisecting GlcNAc depended on the number of residues linked to the 6-positioned mannose. Also, PHN and PMP derivatives produced cross-ring cleavages with abundances higher than observed in the spectra of AB derivatized oligosaccharides. For high-mannose glycans, the most informative cleavages were provided by AB and PHN type of labeling. Here, PMP produced dominant Y-cleavages from the chitobiose while other ions produced weak signals.     

Halogeno-substituted 2-aminobenzoic acid derivatives for negative ion fragmentation studies of N-linked carbohydrates

Negative ion electrospray mass spectra of high-mannose N-linked glycans derivatised with 2-aminobenzoic acids and ionised from solutions containing ammonium hydroxide gave prominent [M-H](-) ions accompanied by weaker [M-2H](2-) ions. Fragmentation of both types of ions gave prominent singly charged glycosidic cleavage ions containing the derivatised reducing terminus and ions from the non-reducing terminus that appeared to be products of cross-ring cleavages. Differentiation of these two groups of ions was conveniently achieved in a single spectrum by use of chloro- or bromo-substituted benzoic acids in order to label ions containing the derivative with an atom with a distinctive isotope pattern. Fragmentation of the doubly charged ions gave more abundant fragments, both singly and doubly charged, than did fragmentation of the singly charged ions, but information of chain branching was masked by the appearance of prominent ions produced by internal cleavages.     

Collision-induced fragmentation of negative ions from N-linked glycans derivatized with 2-aminobenzoic acid

N-Linked glycans from bovine ribonuclease B, chicken ovalbumin, bovine fetuin, porcine thyroglobulin and human alpha(1)-acid glycoprotein were derivatized with 2-aminobenzoic acid by reductive amination and their tandem mass spectra were recorded by negative ion electrospray ionization with a quadrupole time-of-flight mass spectrometer. Derivatives were also prepared from 2-amino-5-methyl- and 2-amino-4,5-dimethoxybenzoic acid in order to confirm the identity of fragment ions containing the reducing terminus. Major fragments from the [M - H](-) ions from the neutral glycans retained the derivative (Y-type cleavages) and provided information on sequence and branching. Other major fragments were products of A-type cross-ring cleavages giving information on antenna structure. Singly doubly and triply charged ions were formed from sialylated glycans. They produced major fragments by loss of sialic acid and a series of singly charged ions that were similar to those from the neutral analogues. Doubly charge ions were also produced by the neutral glycans and were fragmented to form product ions with one and two charges. Again, the fragment ions with a single charge were similar to those from the singly charged parents, but branching information was less obvious because of the occurrence of more abundant ions produced by multiple cleavages. Detection limits were around 200 fmol (3 : 1 signal-to-noise ratio).     

N-(2-diethylamino)ethyl-4-aminobenzamide derivative for high sensitivity mass spectrometric detection and structure determination of N-linked carbohydrates

N-Linked glycans were derivatised by reductive amination using N-(2-diethylamino)ethyl-4-aminobenzamide (DEAEAB, procainamide) and examined by electrospray mass spectrometry. This derivative ionised primarily by protonation of the tertiary amine group and attachment of an alkali metal to give [M + H + X](2+) ions which were much more abundant that doubly charged ions from glycans derivatised with other aromatic amines. Fragmentation of these ions depended on the nature of the alkali metal (X). Lithium and sodium adducts fragmented to give prominent ions produced by cleavages within the DEAEAB derivative whereas the other adducts produced more abundant ions from fragmentation of the carbohydrate. Elimination of a sugar fragment, usually by cleavage adjacent to GlcNAc or sialic acid, together with a hydrogen atom, produced the most abundant singly charged fragment ions. These ions then formally fragmented by glycosidic cleavages. Potassium, rubidium and caesium adducts produced abundant losses of the alkali metal, but the resulting ions appeared not to undergo extensive fragmentation. Most fragment ions from all of the adducts were singly charged, the remainder being doubly charged. Although the spectra of the [M + X + H](2+) ions were not as informative as those from the singly charged ions from other derivatives, they, nevertheless, provided much valuable information on the structure of these glycans.     

Structural characterization by both positive and negative electrospray ion trap mass spectrometry of oligogalacturonates purified by high-performance anion-exchange chromatography

The off-line coupling of high-performance anion-exchange chromatography to electrospray ion trap mass spectrometry (ESI-IT-MS) is described. Two sets of isocratic conditions were optimised for the semi-preparative purification of oligogalacturonates of degree of polymerisation from 4 to 6 by monitoring eluates with either pulsed amperometric detection or evaporative light scattering detection in the presence of an online Dionex Carbohydrate Membrane Desalter (CMD). In these conditions, purified oligogalacturonate solutions were suitable, without further desalting steps, for infusion ESI-IT-MS experiments. This paper provides some interesting features of positive and negative ESI-IT-multiple MS (MSn) of these acidic oligosaccharides. The spectra acquired in both ion modes show characteristic fragments resulting from glycosidic bond and cross-ring cleavages. Under negative ionization conditions, the fragmentation of the singly-charged [M-H]- ions, as well as the Ci-, and Zi-, fragment ions through sequential MSn experiments, was always dominated by product ions from C- and Z-type glycosidic cleavages. All spectra also displayed 0.2 A-type cross-ring cleavage ions which carry linkage information. Collision-induced dissociation (CID) spectra of sodium-cationized species obtained under positive ionization conditions were more complex. Successive MSn experiments also led to the 0.2 A-type cross-ring cleavage ions observed together with B- and Y-type ions. The presence of the 0.2 A ion series was related to Mr 60 (C2H4O2) losses. Combined with the absence of the Mr 30 (CH2O) and the Mr 90 (C3H6O3) ions, these ions were indicative of 1-4 type glycosidic linkage.     

Collisionally activated dissociation and electron capture dissociation provide complementary structural information for branched permethylated oligosaccharides

Doubly charged sodiated and permethylated linear malto-oligosaccharides ({Glc}6-{Glc}9), branched N-linked glycans (high-mannose type GlcNAc2Man5-9, and complex asialo- and disialylated-biantennary glycans) were analyzed by tandem mass spectrometry using collisionally-activated dissociation (CAD) and "hot" electron capture dissociation (ECD) available in a custom-built ESI FTICR mass spectrometer. For linear permethylated malto-oligosaccharides, both CAD and "hot" ECD produced glycosidic cleavages (B, Y, C, and Z ions), cross-ring cleavages (A- and X-type), and internal cleavages (B/Y and C/Y type) to provide sequence and linkage information. For the branched N-linked glycans, CAD and "hot" ECD provided complementary structural information. CAD generated abundant B and Y fragment ions by glycosidic cleavages, whereas "hot" ECD produced dominant C and Z ions. A-type cross-ring cleavages were present in CAD spectra. Complementary A- and X-type cross-ring fragmentation pairs were generated by "hot" ECD, and these delineated the branching patterns and linkage positions. For example, 0, 4An and 3, 5An ions defined the linkage position of the major branch as the 6-position of the central core mannose residue. The internal fragments observed in CAD were more numerous and abundant than in "hot" ECD spectra. Since the triply charged (sodiated) molecular ion of the permethylated disialylated-biantennary N-linked glycan has relatively high abundance, it was isolated and fragmented in a "hot" ECD experiment and extensive fragment ions (glycosidic and complementary pairs of cross-ring cleavages) were generated to fully confirm the sequence, branching, and linkage assignments for this glycan.     

Fragmentation of N-linked glycans with a matrix-assisted laser desorption/ionization ion trap time-of-flight mass spectrometer

N-Linked glycans were ionized from several matrices with a Shimadzu-Biotech AXIMA-QIT matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer. [M+Na]+ ions were produced from all matrices and were accompanied by varying amounts of in-source fragmentation products. The least fragmentation was produced by 2,5-dihydroxybenzoic acid and the most by alpha-cyano-4-hydroxycinnamic acid and 6-aza-2-thiothymine. Sialic acid loss was extensive but could be prevented by formation of methyl esters. Fragmentation produced typical low-energy-type spectra dominated by ions formed by glycosidic cleavages. MS(n) spectra (n = 3 and 4) were used to probe the pathways leading to the major diagnostic ions. Thus, for example, an ion that was formed by loss of the core GlcNAc residues and the 3-antenna was confirmed as being formed by a B/Y rather than a C/Z mechanism. The proposed structures of several cross-ring cleavage ions were confirmed and it was shown that MS3 spectra could be obtained from as little as 10 fmol of glycan.     

Multiple lithium exchange under lithium cationization of cyclodextrins

Multiple lithium exchange is observed during electrospray ionization of alpha-, beta- and gamma-cyclodextrins from aqueous methanolic solution containing LiOH. Apart from [M + Li](+) and [M + nLi - (n - 1)H](+) ions, abundant multiply lithiated doubly charged ions corresponding to [M + nLi - (n - 2)H](2+) ions were observed. At least six lithium exchanges in alpha-cyclodextrin, seven in beta-cyclodextrin and eight in gamma-cyclodextrin were noted. The propensity of multiply lithiated doubly charged ions is much less in the open-ended maltoheptaose. It appears that during droplet or cluster formation and subsequent desolvation, LiOH trapped in the cavity of cyclodextrin reacts to form multiply lithiated ions. The singly charged [M + Li](+) and doubly charged [M + 2Li](2+) ions fragment by glycosidic cleavages, giving B series of ions, whereas the multiply lithiated ions fragment by cross ring cleavages ((2, 4)A or (O, 2)X) followed by glycosidic cleavage. From the tandem mass spectra, it appears that a maximum of two lithium exchanges occur in one sugar unit in these cyclodextrins.     

Electron Transfer Dissociation of Milk Oligosaccharides

For structural identification of glycans, the classic collision-induced dissociation (CID) spectra are dominated by product ions that derived from glycosidic cleavages, which provide only sequence information. The peaks from cross-ring fragmentation are often absent or have very low abundances in such spectra. Electron transfer dissociation (ETD) is being applied to structural identification of carbohydrates for the first time, and results in some new and detailed information for glycan structural studies. A series of linear milk sugars was analyzed by a variety of fragmentation techniques such as MS/MS by CID and ETD, and MS(3) by sequential CID/CID, CID/ETD, and ETD/CID. In CID spectra, the detected peaks were mainly generated via glycosidic cleavages. By comparison, ETD generated various types of abundant cross-ring cleavage ions. These complementary cross-ring cleavages clarified the different linkage types and branching patterns of the representative milk sugar samples. The utilization of different MS(3) techniques made it possible to verify initial assignments and to detect the presence of multiple components in isobaric peaks. Fragment ion structures and pathways could be proposed to facilitate the interpretation of carbohydrate ETD spectra, and the main mechanisms were investigated. ETD should contribute substantially to confident structural analysis of a wide variety of oligosaccharides.     

Production and fragmentation of negative ions from neutral N-linked carbohydrates ionized by matrix-assisted laser desorption/ionization

Although negative ion fragmentation mass spectra of neutral N-linked carbohydrates (those attached to Asn in glycoproteins) provide much more structural information than spectra recorded in positive ion mode, neutral carbohydrates are reluctant to form negative ions by matrix-assisted laser desorption/ionization (MALDI) unless ionized from specific matrices such as nor-harmane or adducted with anions such as chloride. This paper reports the results of experiments to optimize negative ion formation from adducts of N-linked glycans with respect to ion abundance and fragment ion production. The best results were obtained with 2,4,6-trihydroxyacetophenone (THAP) as the matrix with added ammonium nitrate as the salt providing the anion. This approach is demonstrated to be applicable for a wide range of N-linked glycan structures. Phosphate adducts, analogous to those that are usually encountered in electrospray spectra from N-glycans released by protein N-glycosidase F, were produced by addition of ammonium phosphate to the matrix but in relatively low yield allowing competitive ionization of endogenous anionic compounds leading to complex spectra. Fragmentation of the nitrate adducts, which were formed in higher yield, generally paralleled that seen by collision-induced dissociation following ionization by electrospray, with the first stage of the dissociation being the elimination of the nitrate with a proton from one of the hydroxyl groups of the sugar. The spectra of the resulting [M-H](-) species displayed very specific fragment ions, mainly cross-ring and C-type glycosidic cleavage products, that revealed more structural (linkage and branching) information of the compounds than the mainly glycosidic cleavage products that dominated the positive ion spectra.     

New fragmentation mechanisms in matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry of carbohydrates

The spectra recorded by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) of complex carbohydrates from human milk are presented. Besides ions originating from glycosidic cleavages and from sugar ring fragmentations, these spectra show intense peaks that may be assigned to ions produced by three new fragmentation pathways involving a six-atom rearrangement. These ions, together with the A fragments from sugar ring fragmentations, open the possibility of obtaining a complete mapping of the linkage positions present in the carbohydrates investigated by MALDI-TOF/TOF.     

Ionization and fragmentation of neutral and acidic glycosphingolipids with a Q-TOF mass spectrometer fitted with a MALDI ion source

This paper reports the use of a quadrupole time-of-flight (Q-TOF) mass spectrometer fitted with a matrix-assisted laser desorption/ionization (MALDI) ion source for the analysis of neutral and acidic glycosphingolipids. All compounds gave strong [M + Na]+ ions with 2,5-dihydroxybenzoic acid as the matrix, with no loss of sensitivity with increasing mass as was observed from the corresponding ions produced by electrospray. Neutral glycosphingolipids showed negligible in-source fragmentation but sialylated compounds fragmented by loss of sialic acid. However, these losses were not accompanied by unfocused post-source-decay ions as observed with MALDI-reflectron-TOF instruments. The MS/MS spectra were almost identical to those obtained by electrospray. Fragmentation of all compounds was mainly by glycosidic cleavage to give ions, both with and without the ceramide moiety, which defined the carbohydrate chain sequence. Weak ions which defined the sphingosine chain length and abundant ions, produced by loss of the acyl chain, were present when this chain contained a 2-hydroxy group. The technique was applied to the identification of ceramide-trihexosides present in tissues from mice genetically modified to model one of the glycolipid storage diseases (Fabry disease).     

Isolation of inulin‐type oligosaccharides from Chinese traditional medicine: Morinda officinalis How and their characterization using ESI‐MS/MS

Inulin‐type oligosaccharides with different DP were prepared by size‐exclusion chromatography and purity of each oligosaccharide was determined by HPLC equipped with cyclodextrin‐bond column. The purities of obtained inulin‐type oligosaccharides with different DP were more than 98% by one‐step process. The DP and molecular weight were obtained through ESI‐MS in negative mode. The characterization of the inulin‐type oligosaccharides with different DP was studied by MS/MS spectra obtained by collision‐induced dissociation of molecular ions ([M?H]^?). When the DP was lower, the fragment ions were formed through cross‐ring cleavages of two bonds within the sugar ring and glycosidic cleavages. However, with the increase of DP, the ions resulting from glycosidic cleavages between two sugar residues were predominant.     

Ion trap collisional activation of disulfide linkage intact and reduced multiply protonated polypeptides.

The presence of disulfide linkages in multiply charged polypeptide ions tends to inhibit the formation of structurally informative product ions under conventional quadrupole ion trap collisional activation conditions. In particular, fragmentation that requires two cleavages (i.e., cleavage of a disulfide linkage and a peptide linkage) is strongly suppressed. Reduction of the disulfide linkage(s) by use of dithiothreitol yields parent ions upon electrospray without this complication. Far richer structural information is revealed by ion trap collisional activation of the disulfide-reduced species than from the native species. These observations are illustrated with doubly protonated native and reduced somatosin, the [M + 5H](5+) ion of native bovine insulin and the [M + 4H](4+) and [M + 3H](3+) ions of the B-chain of bovine insulin produced by reduction of the disulfide linkages in insulin, and the [M + 11H](11+) ion of native chicken lysozyme and the [M + 11H](11+) and [M + 14H](14+) ions of reduced lysozyme. In each case, the product ions produced by ion trap collisional activation were subjected to ion/ion proton transfer reactions to facilitate interpretation of the product ion spectra. These studies clearly suggest that the identification of polypeptides with one or more disulfide linkages via application of ion trap collisional activation to the multiply charged parent ions formed directly by electrospray could be problematic. Means for cleaving the disulfide linkage, such as reduction by dithiothreitol prior to electrospray, are therefore desirable in these cases.     

Assignment of acetyl groups to O-2 and/or O-3 of pectic oligogalacturonides using negative electrospray ionization ion trap mass spectrometry

Partially acetylated and methylated oligogalacturonides produced by enzymatic hydrolysis of sugar beet pectin were analysed by negative electrospray ionization ion trap mass spectrometry (ESI-ITMS). The (18)O labelling of the oligomer reducing end allowed the precise assignment of the fragments resulting from glycosidic bond and cross-ring cleavages. The collisional-induced dissociation of the C(i) and Z(j) fragment ions through sequential MS(n) experiments always displayed (0, 2)A-type cross-ring cleavage ions which were related to C(2)H(4)O(2) losses. These (0, 2)A ions appeared to be highly diagnostic ions allowing the precise location of the acetyl groups to the O-2 and/or O-3 of the acetylated galacturonic acid residues.     

Competing fragmentation processes in tandem mass spectra of heparin-like glycosaminoglycans

Heparin-like glycosaminoglycans (HLGAGs) are highly sulfated, linear carbohydrates attached to proteoglycan core proteins and expressed on cell surfaces and in basement membranes. These carbohydrates bind several families of growth factors and growth factor receptors and act as coreceptors for these molecules. Tandem mass spectrometry has the potential to increase our understanding of the biological significance of HLGAG expression by providing a facile means for sequencing these molecules without the need for time-consuming total purification. The challenge for tandem mass spectrometric analysis of HLGAGs is to produce abundant ions derived via glycosidic bond cleavages while minimizing the abundances of ions produced from elimination of the fragile sulfate groups. This work describes the competing fragmentation pathways that result from dissociation of high negative charge state ions generated from HLGAGs. Glycosidic bond cleavage ion formation competes with losses of equivalents of H2SO4, resulting in complex ion patterns. For the most highly sulfated structure examined, an octasulfated tetramer, an unusual loss of charge from the precursor ion was observed, accompanied by low abundance ions originating from subsequent backbone cleavages. These results demonstrate that fragmentation processes competing with glycosidic bond cleavages are more favored for highly sulfated HLGAG ions. In conclusion, reduction of charge-charge repulsions, such as is achieved by pairing the HLGAG ions with metal cations, is necessary in order to minimize the abundances of ions derived via fragmentation processes that compete with glycosidic bond cleavages.     

Determination of linkage position and anomeric configuration in Hex-Fuc disaccharides using electrospray ionization tandem mass spectrometry

Fixed-energy sequential tandem mass spectrometry (MS(n)) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of six isomers of Gal-Fucalpha-OBenzyl disaccharides. Under collision-induced dissociation (CID), sodiated molecular species generated in the positive-ion electrospray ionization mode yield simple and predictable mass spectra. Information on interglycosidic linkages and configurations can be deduced from the relative intensities of the selected diagnostic fragments arising from the glycosidic bond cleavages and corroborated by the fragments arising from cross-ring cleavages. As the CID patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by matching the spectra with a library. The rules governing the fragmentation behavior of this class of oligosaccharides were tested for a representative isomeric disaccharide, Glcbeta1,3Fucalpha-OAllyl. The findings establish a basis for using MS(n) with a quadrupole ion trap instrument to elucidate structures of hexose-fucose subunits from more complicated oligosaccharides. Energy-resolved mass spectra were also acquired by CID tandem triple-quadrupole mass spectrometry. The breakdown behavior of the molecular ions revealed patterns which could differentiate stereoisomers of Gal-Fuc disaccharides over a range of collision energy from 20 to 50 eV.     

Collision-induced fragmentation of underivatized N-linked carbohydrates ionized by electrospray

The electrospray mass spectra and collision-induced fragmentation of neutral N-linked glycans obtained from glycoproteins were examined with a Q-TOF mass spectrometer. The glycans were ionized most effectively as adducts of alkali metals, with lithium providing the most abundant signal and caesium the least. Singly charged ions generally gave higher ion currents than doubly charged ions. Addition of formic acid could be used to produce [M + H]+ ions, but these ions were always accompanied by abundant cone-voltage fragments. The energy required for collision-induced fragmentation was found to increase in a linear manner as a function of mass with the [M + Na]+ ions requiring about four times as much energy as the [M + H]+ ions for complete fragmentation of the molecular ions. Fragmentation of the [M + H]+ ions gave predominantly B- and Y-type glycosidic fragments whereas the [M + Na]+ and [M + Li]+ ions produced a number of additional fragments including those derived from cross-ring cleavages. Little fragmentation was observed from the [M + K]+ and [M + Rb]+ ions and the only fragment to be observed from the [M + Cs]+ ion was Cs+. The [M + Na]+ and [M + Li]+ ions from all the N-linked glycans gave abundant fragments resulting from loss of the terminal GlcNAc moiety and prominent, though weaker, ions as the result of 0,2A and 2,4A cross-ring cleavages of this residue. Most other ions were the result of successive additional losses of residues from the non-reducing terminus. This pattern was particularly prominent with glycans containing several non-reducing GlcNAc residues where successive losses of 203 u were observed. Many of the ions in the low-mass range were products of several different fragmentation routes but still provided structural information. Possibly of most diagnostic importance was an ion formed by loss of 221 u (GlcNAc molecule) from an ion that had lost the 3-antenna and the chitobiose core. This latter ion, although coincident in mass with some other 'internal' fragments, often provided additional information on the composition of the antennae. Other ions defining antenna composition were weak cross-ring fragments produced from the core branching mannose residue. Glycans containing Gal-GlcNAc residues showed successive losses of this moiety, particularly from the B-type fragments resulting from loss of the reducing-terminal GlcNAc residue. The [M + Na]+ and [M + Li]+ ions from high-mannose and hybrid glycans gave a series of ions of composition (Man)nNa/Li+ where n = 1 to the total number of glycans in the molecule, allowing these sugars to be distinguished from the more highly processed complex glycans. Other ions in the spectra of the high-mannose glycans were diagnostic of chain branching but insufficient information was available to determine their mode of formation.