期刊
JOURNAL OF PROTEOME RESEARCH
卷 8, 期 2, 页码 556-566出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr800734r
关键词
glycoproteins; mass spectrometry; proteomics; plasma; proteomic reactor; microscale
资金
- Genome Canada through the Ontario Genomics Institute [2008-OGI-TD-01]
- Province of Ontario
- University of Ottawa
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 O-18-labeling and deglycosylation, alkylation, tryptic digestion and pH based fractionation in a device that has an interstitial volume (reaction volume) of similar to 1 mu L. 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 mu L 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.
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