Ionisation and fragmentation of complex glycans with a quadrupole time‐of‐flight mass spectrometer fitted with a matrix‐assisted laser desorption/ionisation ion source

A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b-and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 16 . This requires only small amounts of protein, provides a relatively fast identification and can easily be automated. In the second approach, fragment ion masses of one or more proteolytic peptides from a protein digest are determined, usually by electrospray ionization tandem mass spectrometry (ESI MS/MS). Database searches can then be performed with different algorithms; either by using partly interpreted data or by submitting the list of fragment ions taken from the MS/MS spectra without any data interpretation [2,3]. However, both approaches suffer from some limitations. In peptide mass fingerprinting, results obtained are not always unambiguous and the method does not cope well with protein mixtures. On the other hand,

Section: Maldi-q-tof Set-upmentioning

confidence: 99%

Sequence dependent fragmentation of peptides generated by MALDI quadrupole time-of-flight (MALDI Q-TOF) mass spectrometry and its implications for protein identification

Wattenberg

Organ

Schneider

et al. 2002

“…Different combinations of ionization techniques, ion activation, and mass analyzers have been used, including fast atom bombardment (FAB)-MS [10 -12], infrared-laser desorption MS [13,14], matrix-assisted laser desorption/ionization (MALDI)/magnetic sector MS [15][16][17], electrospray ionization (ESI)-MS [18,19], MALDI/time-offlight (TOF)-MS [20 -22], ESI ion-trap MS [23][24][25], MALDI Fourier transform MS [26], ESI-or MALDIbased quadrupole/TOF-MS [27][28][29][30], MALDI/Postsource Decay (PSD) TOF-MS [31][32][33], and, more recently, MALDI-TOF/TOF-MS [34 -38].…”

mentioning

confidence: 99%

Identification of isomeric N-glycan structures by mass spectrometry with 157 nm laser-induced photofragmentation

Devakumar

Mechref

Kang

et al. 2008

Characterization of structural isomers has become increasingly important and extremely challenging in glycobiology. This communication demonstrates the capability of ion-trap mass spectrometry in conjunction with 157 nm photofragmentation to identify different structural isomers of permethylated N-glycans derived from ovalbumin without chromatographic separation. The results are compared with collision-induced dissociation (CID) experiments. Photodissociation generates extensive cross-ring fragment ions as well as diagnostic glycosidic product ions that are not usually observed in CID MS/MS experiments. The detection of these product ions aids in characterizing indigenous glycan isomers. The ion trap facilitates MS n experiments on the diagnostic glycosidic fragments and cross-ring product ions generated through photofragmentation, thus allowing unambiguous assignment of all of the isomeric structures associated with the model glycoprotein used in this study. Photofragmentation is demonstrated to be a powerful technique for the structural characterization of glycans. , and protein regulations and interactions [6], is widely acknowledged [7]. To improve our understanding of these processes, it is important to explore glycan structure-function relationships. However, the detailed characterization of a glycan structure and its attributes remains a difficult task, given the microheterogeneity and diversity of these molecules. Importantly, the discrimination of numerous structural isomers differing in sequence, linkage, position, or branching features remains a challenging frontier of glycobiology.For years, the characterization of glycan structures has almost exclusively been accomplished by tandem mass spectrometry (MS/MS) [1,8,9] because of its high sensitivity and minimum sample requirements relative to nuclear magnetic resonance (NMR). Different combinations of ionization techniques, ion activation, and A glycan ion generally fragments in two ways: (1) glycosidic cleavages resulting from a bond rupture between two adjacent sugar residues and (2) cross-ring cleavages in which any two bonds on the same sugar unit are broken. Cross-ring fragment ions are commonly observed in high-energy collision-induced dissociation (CID) methods as demonstrated in the tandem TOF/TOF approach [34 -38]. A limitation of this technique, however, is its inability to perform multistage tandem mass spectrometry experiments. On the other hand, glycosidic cleavage ions are predominantly observed in low-energy activation methods and are mainly used to derive sequence and limited branching information. Most recently, demonstrated the use of low-energy activation to identify the structural isomers of glycans in complex mixtures with the help of sequential tandem mass spectrometry (MS n ). However, a disadvantage of CID is the decrease in both the degree and efficiency of dissociation with increasing mass and MS n events. Alternatively, other activation techniques have been used for Address reprint requests to Prof. James P.

“…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.…”

mentioning

confidence: 99%

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

Harvey

2005

Self Cite

246

189

Negative ion spectra of N-linked glycans were produced by electrospray from a dilute solution of the glycans and various salts in methanol:water using a Waters-Micromass Q-TOF Ultima Global tandem quadrupole/time-of-flight (Q-TOF) mass spectrometer. Stable anionic adducts were formed with chloride, bromide, iodide, nitrate, sulphate, and phosphate. Unstable adducts that fragmented by a cross-ring cleavage of the reducing N-acetylglucosamine (GlcNAc) residue, were formed with fluoride, nitride, sulphide, carbonate, bicarbonate, hydroxide, and acetate. Nitrate adducts prepared from ammonium nitrate produced the most satisfactory spectra as they were relatively free from in-source fragmentation products and gave signals that were about ten times as strong as those from corresponding [M Ϫ H] Ϫ ions prepared from solutions containing ammonium hydroxide. Detection limits were in the region of 20 fmol. Neutral glycans gave both singly-and doubly-charged ions with the larger glycans preferring the formation of doubly-charged ions. Acidic glycans with several acidic groups gave ions in higher charge states as the result of ionization of the anionic groups. Low energy collision-induced decomposition (CID) spectra of the singly-charged ions were dominated by cross-ring and C-type fragments, unlike the corresponding spectra of the positive ions that contained mainly B-and Y-type glycosidic fragments. Formation of these ions could be rationalized by proton abstraction from various hydroxy groups by an initially-formed anionic adduct. Prominent glycosidic and cross-ring cleavage ions defined structural features such as the specific composition of each of the two antennae, presence of a bisecting GlcNAc residue and location of fucose residues, details that were difficult to determine by conventional techniques. Acidic glycans fragmented differently on account of charge localization on the acid functions rather than the hydroxy groups. (J Am Soc Mass Spectrom 2005, 16, 622-630)