Glycoproteomic Reactor for Human Plasma
journal contributionposted on 06.02.2009, 00:00 by Hu Zhou, Weimin Hou, Nicholas J. Denis, Houjiang Zhou, Julian Vasilescu, Hanfa Zou, Daniel Figeys
We describe the development of a glycoproteomic reactor that combines multiple biochemical and chemical protein processing into a single device for the study of N-glycosylated proteins. The glycoproteins are first enriched by concanavalin A affinity chromatography and then transferred onto and efficiently processed in the glycoproteomic reactor. This glycoproteomic reactor combines protein concentration and purification, disulfide bond reduction, peptide-N-glycosidase-mediated 18O-labeling and deglycosylation, alkylation, tryptic digestion and pH based fractionation in a device that has an interstitial volume (reaction volume) of ∼1 μL. We demonstrated the potential of the glycoproteomic reactor using human plasma. Under stringent criteria, 82 unique glycopeptides representing 41 unique glycoproteins were identified from as little as 5 μL of human plasma. Our glycoproteomic reactor reduces the sample processing time to less than 1.5 h, reduces the reagent consumption while providing over 1000-fold concentration of the sample, provides efficient removal of high concentration of glycan buffer, and, finally, allows both glycopeptides and nonglycosylated tryptic peptides to be analyzed by the mass spectrometer which provides much greater protein coverage and more reliable identifications.