N-linked oligosaccharide analysis of rat brain Thy-1 by liquid chromatography with graphitized carbon column/ion trap-Fourier transform ion cyclotron resonance mass spectrometry in positive and negative ion modes.
We have previously described the site-specific glycosylation analysis of rat brain Thy-1 by LC/multistage tandem mass spectrometry (MS(n)) using proteinase-digested Thy-1. In the present study, detailed structures of oligosaccharides released from Thy-1 were elucidated by mass spectrometric oligosaccharide profiling using LC/MS with a graphitized carbon column (GCC-LC/MS). First, using model oligosaccharides, we improved the oligosaccharide profiling by ion trap mass spectrometry (IT-MS) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Sequential scanning of a full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in positive ion mode, and a subsequent full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in negative ion mode enabled the monosaccharide composition analysis as well as profiling and sequencing of both neutral and acidic oligosaccharides in a single analysis. The improved oligosaccharide profiling was applied to elucidation of N-linked oligosaccharides from Thy-1 isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was demonstrated that Thy-1 possesses a significant variety of N-linked oligosaccharides, including Lewis a/x, Lewis b/y, and disialylated structure as a partial structure. Our method could be applicable to analysis of a small abundance of glycoproteins, and could become a powerful tool for glycoproteomics.
Itoh
Satsuki
S
Division of Biological Chemistry and Biologicals, National Institute of Health Science, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Kawasaki
Nana
N
Hashii
Noritaka
N
Harazono
Akira
A
Matsuishi
Yukari
Y
Hayakawa
Takao
T
Kawanishi
Toru
T
D000818:Animals; D001923:Brain Chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D017357:Cyclotrons; D005583:Fourier Analysis; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D025942:Oligosaccharides, Branched-Chain / Q000032:analysis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D051381:Rats; D018800:Thy-1 Antigens / Q000737:chemistry*
D025942:Oligosaccharides, Branched-Chain; D018800:Thy-1 Antigens; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase