High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification.
Protein glycosylation is an important post-translational modification associated, among others, with diseases and the efficacy of biopharmaceuticals. Matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (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 α2,3- and α2,6-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 α2,6-linked sialic acids and lactonization of α2,3-linked variants, in short time using mild conditions. Glycans were recovered by hydrophilic interaction liquid chromatography solid phase extraction and analyzed by MALDI-TOF-MS in reflectron positive mode with 2,5-dihydroxybenzoic acid as the 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.
Center for Proteomics and Metabolomics, Leiden University Medical Center , 2300 RC Leiden, The Netherlands.
Karli R
KR
Reiding
Dennis
D
Blank
Dennis M
DM
Kuijper
André M
AM
Deelder
Manfred
M
Wuhrer
D004951:Esterification; D006031:Glycosylation; D057166:High-Throughput Screening Assays; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D011506:Proteins; D019158:N-Acetylneuraminic Acid