Highly sensitive multistage mass spectrometry enables small-scale analysis of protein glycosylation from two-dimensional polyacrylamide gels

Electrophoresis
Nobuaki TakemoriHiroyuki Matsumoto

Abstract

Structural characterization of glycoproteins remains among the most challenging areas of glycomics due to the requirement of large quantities of samples and laborious biochemical steps involved in the analytical procedure. Here we report the structural characterization of glycoproteins separated on a 2-D gel by using a MALDI-QIT-TOF MS where QIT is quadrupole IT. The combination of MALDI-ion source and QIT appears to generate a unique tendency to cause fragmentation of glycopeptides without collision-induced dissociation. The majority of such fragmentations observed in our study result from the cleavage of sugar linkages, but not of peptide-peptide or peptide-sugar linkages. This unique feature allows us to perform pseudo-MS3 analysis of a fragmented glycopeptide. A small gel spot of a glycoprotein in the abundance range of low picomoles was enough for the mass spectrometer to analyze fragmentation pathway of the sugar linkage and peptide backbone. In this study, we demonstrate direct determination of glycosylation sites and N-linked glycan-sequences of the tryptic glycopeptides of Drosophila glycoproteins. Glycopeptides with various MWs up to approximately 4000 Da were suitable for structural analysis, including its attachment...Continue Reading

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Citations

Aug 26, 2010·Molecular & Cellular Proteomics : MCP·Sheng PanTeresa A Brentnall
Nov 16, 2011·International Journal of Proteomics·Evelyn H Kim, David E Misek
Jun 30, 2009·Analytical Biochemistry·Harumi OsagoMikako Tsuchiya
Oct 20, 2006·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Manfred WuhrerCornelis H Hokke
Jan 3, 2007·Journal of Mass Spectrometry : JMS

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