Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins

Patel, T.P.; Bruce, Jim; Merry, Anthony H.; Bigge, Collette; Wormald, Mark R.; Jaques, A J; Parekh, Raj B.

doi:10.1021/bi00053a037

Cited by 370 publications

(230 citation statements)

References 38 publications

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“…N-linked glycans were released by large scale hydrazinolysis followed by reacetylation [39,40] from the well-characterized glycoproteins (Sigma Chemical Co. Ltd., Poole, Dorset, UK) ribonuclease B [41], porcine thyroglobulin [42,43], chicken ovalbumin [44,45], and bovine fetuin [46]. Methanol was obtained from BDH Ltd. (Poole, UK) and the various inorganic salts were from Aldrich Chemical Co. Ltd. (Poole, UK) and BDH.…”

Section: Methodsmentioning

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

J. Am. Soc. Mass Spectrom.

244

189

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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)

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Section: Methodsmentioning

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

J. Am. Soc. Mass Spectrom.

244

189

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“…This method has worked well in our hands. Other methods, including gel reductive b-elimination, have been described [16][17][18][19] .…”

Section: Release Of Glycansmentioning

confidence: 99%

Analysis of protein glycosylation by mass spectrometry

2007

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We present a detailed protocol for the structural analysis of protein-linked glycans. In this approach, appropriate for glycomics studies, N-linked glycans are released using peptide N-glycosidase F and O-linked glycans are released by reductive alkaline b-elimination. Using strategies based on mass spectrometry (matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano-electrospray ionization mass spectrometry/mass spectrometry (nano-ESI-MS-MS)), chemical derivatization, sequential exoglycosidase digestions and linkage analysis, the structures of the N-and/or O-glycans are defined. This approach can be used to study the glycosylation of isolated complex glycoproteins or of numerous glycoproteins encountered in a complex biological medium (cells, tissues and physiological fluids).

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“…N-Linked glycans were released from glycoproteins by hydrazinolysis. 34 Briefly, anhydrous hydrazine was added to dried glycoprotein samples, heated to 100 °C for 6 h, and then evaporated under a nitrogen stream. The bulk sample was re-N-acetylated in saturated sodium bicarbonate with acetic anhydride, desalted on Dowex 50W cation-exchange resin, and purified by cellulose column chromatography 18 (slurry packed with medium fibrous cellulose in 1-butanol/ethanol/water, 4:1:1).…”

Section: Sample Preparationmentioning

confidence: 99%

“…6 In both cases, however, the product structures remain to be characterized. A large body of work has shown that spectral comparisons of isomers at different stages of sequential molecular fragmentation (MS n ) [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] are clearly different, although in one case, reproducibility has been reported to be "vague". 25 In our experience, spectral reproducibility has been excellent, 26 and this very feature has provided an opportunity to build a searchable fragment library 27 and derive glycan structure with supporting tools.…”

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confidence: 99%

Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

et al. 2007

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Consistent with the goals of a comprehensive carbohydrate sequencing strategy, we extend earlier reports to include the characterization of structural (constitutional) isomers. Protocols were developed around ion trap instrumentation providing sequential mass spectrometry (MS n ) and supported with automation and related computational tools. These strategies have been built on the principle that for a single structure all product spectra upon sequential fragmentation are reproducible and each stage represents a rational spectrum of its precursor; i.e., all major fragments should be accounted for. Anomalous ions at any stage are clues indicating the presence of structural isomers. Gas-phase isolation and subsequent fragmentation of such ions provide an opportunity to specifically resolve selected structures for their detailed characterization. Importantly, some isomers were not detected following MS 2 and required multiple (MS n>2 ) stages for their characterization. Derivatization remains critical to position substructures in a glycan array since product ions carry fragmentation "scars" throughout the MS n tree. Equally as important are the pathway relationships between each stage and the greater yield of fragments with the smaller number of oscillators. Applications were directed to the structural isomers in ovalbumin and IgG, where, in the latter case, several previously unreported glycans were detected. Procedures were supported with bioinformatics tools for assimilating structure from the MS n data sets.A comprehensive carbohydrate sequence is particularly challenging when considering the constitutional and stereoisomers that are abundant in glycan arrays. Constitutional isomers, also referred to as structural isomers, are defined as an identical atomic composition arranged in a different structure. Stereoisomers are exemplified with those frequently encountered in the hexoses, mannose, galactose, and glucose, while the two G1 glycans found in IgG are representative of simple structural isomers. In this latter case, an identical monomer may be linked on either of two antennae providing a different topology. In another nomenclature clarification, the term isomer is often confused with isobar. which relates to a different composition of atoms occurring at a nominal (unit) mass resolution. 1 At the MS profile stage, isobars are not usually a significant problem in carbohydrate structural characterization, but structural isomers and stereoisomers are. During fragmentation, however, isobaric fragments may be formed, but identification will require higher mass resolving power and mass accuracy.

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Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins

Cited by 370 publications

References 38 publications

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

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

Analysis of protein glycosylation by mass spectrometry

Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

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