Sialic acid-focused quantitative mouse serum glycoproteomics by multiple reaction monitoring assay.
Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model.
Kurogochi
Masaki
M
Graduate School of Life Science, Frontier Research Center for the Post-Genomic Science and Technology, Hokkaido University, Kita-ku, Sapporo, Japan.
Matsushista
Takahiko
T
Amano
Maho
M
Furukawa
Jun-ichi
J
Shinohara
Yasuro
Y
Aoshima
Masato
M
Nishimura
Shin-Ichiro
S
D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000379:methods; D003920:Diabetes Mellitus / Q000097:blood; D004195:Disease Models, Animal; D005260:Female; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
D006020:Glycopeptides; D019158:N-Acetylneuraminic Acid