Document Detail

High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification.
MedLine Citation:
PMID:  24831253     Owner:  NLM     Status:  Publisher    
Protein glycosylation is an important post-translational modification associated, among others, with diseases and the efficacy of biopharmaceuticals. MALDI-TOF-MS can be performed to study glycosylation in a high-throughput manner, but is hampered by the instability and ionization bias experienced by sialylated glycan species. Stabilization and neutralization of these sialic acids can be achieved by permethylation or by specific carboxyl group derivatization with the possibility of discrimination between alpha2,3- and alpha2,6-linked sialic acids. However, these methods typically require relatively pure glycan samples, show sensitivity to side reactions, and need harsh conditions or long reaction times. We established a rapid, robust and linkage-specific high-throughput method for sialic acid stabilization and MALDI-TOF-MS analysis, to allow direct modification of impure glycan-containing mixtures such as PNGase F-released human plasma N-glycome. Using a combination of carboxylic acid activators in ethanol achieved near-complete ethyl esterification of alpha2,6-linked sialic acids and lactonization of alpha2,3-linked variants, in short time using mild conditions. Glycans were recovered by HILIC SPE and analyzed by MALDI-TOF-MS in reflectron positive mode with 2,5-dihydroxybenzoic acid as matrix substance. Analysis of the human plasma N-glycome allowed high-throughput detection and relative quantitation of more than 100 distinct N-glycan compositions with varying sialic acid linkages.
Karli Robert Reiding; Dennis Blank; Dennis M Kuijper; André M Deelder; Manfred Wuhrer
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2014-5-15
Journal Detail:
Title:  Analytical chemistry     Volume:  -     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2014 May 
Date Detail:
Created Date:  2014-5-16     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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