Robust and high-throughput sample preparation for (semi-)quantitative analysis of N-glycosylation profiles from plasma samples.
High-throughput quantitative or semiquantitative glycoanalytical procedures are urgently required for biomarker discovery and quality control of glycoprotein pharmaceuticals. We describe here a strategy for high-throughput sample preparation for the relative quantitation of N-glycan patterns of human plasma glycoproteins. To this end, efficient glycan release is combined with a robust labeling step. Sample purification is achieved using solid phase extraction with a hydrophilic interaction chromatography stationary phase, which allows the separation of the labeled glycans from excess label, proteins, and matrix constituents such as salts and lipids. We show that the obtained labeled glycan sample solution is directly compatible with a number of downstream analytical techniques such as hydrophilic interaction and reverse-phase chromatography, capillary electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry. High throughput is achieved by conducting all sample preparation steps in the 96-well format. The robustness and repeatability of the sample preparation strategy is evaluated.
Department of Parasitology, Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, Leiden, The Netherlands.
L Renee
LR
Ruhaak
Carolin
C
Huhn
Carolien A M
CA
Koeleman
André M
AM
Deelder
Manfred
M
Wuhrer
D000925:Anticoagulants / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D019343:Citric Acid / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D013058:Mass Spectrometry; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D013194:Staining and Labeling; D062367:ortho-Aminobenzoates / Q000737:chemistry
D000925:Anticoagulants; D001798:Blood Proteins; D007202:Indicators and Reagents; D009844:Oligosaccharides; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D019343:Citric Acid