A New Analytical Method for Glycoprotein Structure Analysis Using MALDI-QIT-TOFMS: An Application to Ribonuclease B

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Abstract
Mass spectrometry (MS) is becoming an indispensable analytical tool for glycoproteomics, that is, for the analysis of glycan and protein structures of glycoproteins. While a simple and rapid MS method which can minimize troublesome procedures for sample preparation is desired, a single mass spectrometer to meet this demand has not been realized to date. We report here a procedure for the structure analysis of Ribonuclease B (RNase B) using the MS and MSn analyses of protonated ions ([M+H]+) and sodium-adducted ions ([M+Na]+) by a single MALDI-QIT-TOFMS. Five peaks were identified as a glycopeptide in mass spectra with 2,5-dihydroxybenzoic acid (DHBA) as a matrix for RNase B digests by Lysyl endopeptidase (Lys C) without desalting. All of the five [M+H]+ peaks were accompanied by [M+Na]+ peaks. The same fragment peaks were observed in low mass area of all MS/MS spectrum for each [M+H]+ or [M+Na]+ species. MSn analyses using [M+H]+ as a precursor ion provided the information of peptide sequence and glycosylation site, while fragments from [M+Na]+ showed oligosaccharide sequence. Finally, this technique using [M+H]+ and [M+Na]+ with MALDI-QIT-TOFMS realized an easy and high-throughput implementation of glycoprotein structure analyses.

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