Collision-induced fragmentation of underivatizedN-linked carbohydrates ionized by electrospray

Abstract

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.

Fragmentation of negative ions from carbohydrates: Part 1. Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from N-linked carbohydrates

“…Compared with the [M ϩ Na] ϩ ions in the MALDI spectrum, where previous work has shown that glycans across this mass range all produce comparable signals [55],°the°[M°ϩ°NO 3 ] Ϫ ions from the neutral glycans showed a slight drop in sensitivity with increasing mass but to a far lesser extent than the [M ϩ Na] ϩ ions produced°by°electrospray°(Figure°1d)° [23].°The°signal strength from the larger glycans was stronger than that from any of the other adducts in either positive or negative ion modes and the spectra were less complicated, particularly compared with the MALDI spectrum concentration against signal strength were linear from about 20 pmol/ L (100 pmol infused) to the detection limit of about 20 fmol infused (3:1 signal:noise). Copper and potassium nitrate readily formed salt clusters such that to record the signals from the lower concentrations of glycan, the amount of nitrate added to the electrospray solution had to be reduced in order to minimise cluster formation to acceptable levels.…”

“…[M ϩ Na] ϩ Ions can be obtained, as in MALDI, and reach their highest abundance at high cone voltages [23]. However, they are usually accompanied by a considerable amount of in-source fragmentation.…”

“…Sensitivity generally falls as a function of increasing mass such that it is difficult to record [M ϩ Na] ϩ ions from the larger glycans. Nevertheless, both the [M ϩ Na] ϩ ions from underivatized and derivatized glycans and ions formed by adduction with other metals provide informative spectra containing both glycosidic and cross-ring fragments [22][23][24][25][26][27][28]. Both [M ϩ H] ϩ and doubly-charged ions produced by protonation are formed under milder ion-source conditions but their MS/MS spectra are comparatively simple and do not generally contain ions produced by cross-ring cleavages.…”

Analysis of protein glycosylation by mass spectrometry

“…This instrument is used to obtain fragmentation spectra in a short time with high mass accuracy that reveal detailed structural and in some cases linkage information on complex oligosaccharides. Fragmentation of carbohydrates is dependent on several factors, such as the type of ion formation, its charge state and the energy deposited into the ion 29,30 the sugar rings and involves a hydrogen migration, and cross-ring cleavages that involve the rupture of two bonds on the same sugar residue. The nomenclature generally used for describing these fragment ions is that proposed by Domon and Costello 31 .…”

0,2An cross-ring cleavage as a general diagnostic tool for glycan assignment in glycoconjugate mixtures

“…, but an assignment of a carbohydrate structure from a single experiment still represents a major task. A number of techniques has been applied for structural elucidation of components in carbohydrate mixtures, basically attempting to find diagnostic fragment ions responsible for single structures [2][3][4][5][6][7][8][9][10][11][12][13]. The possibility to obtain distinct fragmentation patterns under controlled collision energy and gas conditions along with the ability of automatic switching between MS and MS/MS mode by electrospray quadrupole time-of-flight Q-TOF instrument, represents a powerful option for high-throughput analysis of complex carbohydrate mixtures.…”