Rapid Identification of Terminal Sialic Acid Linkage Isomers by Pseudo-MS super(3) Mass Spectrometry
We used pseudo-triple-stage tandem MS to rapidly differentiate terminal alpha (2,3)- and alpha (2,6)-linked sialic acids of glycopeptides and native glycans. The precursor ions generate important diagnostic fragments to distinguish different terminal linked isomeric glycans and glycopeptides based o...
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Published in | Israel journal of chemistry Vol. 55; no. 3-4; pp. 412 - 422 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
01.04.2015
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Subjects | |
Online Access | Get full text |
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Summary: | We used pseudo-triple-stage tandem MS to rapidly differentiate terminal alpha (2,3)- and alpha (2,6)-linked sialic acids of glycopeptides and native glycans. The precursor ions generate important diagnostic fragments to distinguish different terminal linked isomeric glycans and glycopeptides based on collision-induced dissociation (CID). The diagnostic ions include hexose linked with N-acetylhexosamine residues, sialic acid with the loss of water, and sialic acid residues. Their values of mass-to-charge ratio (m/z) are at 366.14, 274.09, and 292.10, respectively. The relative intensity ratios (RIR) were calculated by the intensity of signals at m/z 274.09 and 292.10 divided by the intensity of the signal at m/z 366.14. Under optimal conditions of an in-source fragmentation voltage of 70V and a normalized collision energy of 30%, the difference in RIR of diagnostic ions between terminal alpha (2,3)-linked sialic acid of glycopeptide (1.3) and terminal alpha (2,6)-linked sialic acid of glycopeptide (<0.18) is the largest. Moreover, quantitative calibration curves with different percentages of alpha (2,3)- and alpha (2,6)-linked sialoglycopeptides demonstrated that this method applied not only to biantennary N-linked glycan, but also to complex and hybrid N-linked glycans and O-linked glycans. From measurements of the RIR of diagnostic ions, erythropoietin, fetuin, and herceptin have 98, 70, and 100%, respectively, alpha (2,3)-linked N-sialic acid of total terminal sugar moieties. N-Glycosylation of the antibody-binding fragment (Fab) moiety of cetuximab has 100% alpha (2,6)-linked N-glycolylneuraminic acid terminal glycan linkages. This method can be applied to identify compounds from chemical synthesis. It can also be used for quality evaluation of bio-similar protein drugs. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 content type line 23 ObjectType-Feature-2 |
ISSN: | 0021-2148 1869-5868 |
DOI: | 10.1002/ijch.201400141 |