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An integrated strategy for high-sensitive and multi-level glycoproteome analysis from low micrograms of protein samples

ABSTRACT: We describe here a simple and integrated spintip-based glycoproteomics technology,termed Glyco-SISPROT, for challenging the lowest starting material limitation and achieving a comprehensive view of glycoproteome with shortest sample processing time. By carefully integrating and optimizing SCX, C18 and Concanavalin A packing material and their combination in spintip format, both predigested peptides and protein lysates could be processed by Glyco-SISPROT with high efficiency. More importantly, deglycopeptide, intact glycopeptide and glycans released by multiple glycosidases could be readily collected from the same Glyco-SISPROT workflow for LC-MS analysis. In total, above 1850 glycosites in 1770 unique deglycopeptides were characterized from mouse liver by using either 100 macrograms of predigested peptides or directly using 100 macrograms of protein lysates, in which about 30 percentage of glycosites were released by both PNGase F and Endos. To the best of our knowledge, this approach should be one of the most comprehensive glycoproteomic approaches by using limited protein starting material. One significant benefit of Glyco-SISPROT was that whole processing time was dramatically reduced from a few days to only 6 hours with good reproducibility when protein lysates were directly processed by Glyco-SISPROT.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Ruijun Tian

PROVIDER: MSV000083579 | MassIVE | Fri Mar 15 04:52:00 GMT 2019

REPOSITORIES: MassIVE

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An integrated strategy for high-sensitive and multi-level glycoproteome analysis from low micrograms of protein samples

Project description:Glycosylation, as a biologically important protein post-translational modification, often alters on both glycosites and glycans, simultaneously. However, most of current approaches focused on biased profiling of either glycosites or glycans, and limited by time-consuming process and milligrams of starting protein material. We describe here a simple and integrated spintip-based glycoproteomics technology (termed Glyco-SISPROT) for achieving a comprehensive view of glycoproteome with shorter sample processing time and low microgram starting material. By carefully integrating and optimizing SCX, C18 and Concanavalin A (Con A) packing material and their combination in spintip format, both predigested peptides and protein lysates could be processed by Glyco-SISPROT with high efficiency. More importantly, deglycopeptide, intact glycopeptide and glycans released by multiple glycosidases could be readily collected from the same Glyco-SISPROT workflow for LC-MS analysis. In total, above 1850 glycosites in ~1770 unique deglycopeptides were characterized from mouse liver by using either 100 microgram of predigested peptides or directly using 100 microgram of protein lysates, in which about 30 percentage of glycosites were released by both PNGase F and Endos. To the best of our knowledge, this approach should be one of the most comprehensive glycoproteomic approaches by using limited protein starting material. One significant benefit of Glyco-SISPROT is that whole processing time is dramatically reduced from a few days to less than 6 hours with good reproducibility when protein lysates were directly processed by Glyco-SISPROT. We expect that this method will be suitable for multi-level glycoproteome analysis of rare biological samples with high sensitivity.

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