Enhanced analysis of the mouse plasma proteome using cysteine-containing tryptic glycopeptides.
A comprehensive understanding of the mouse plasma proteome is important for studies using mouse models to identify protein markers of human disease. To enhance our analysis of the mouse plasma proteome, we have developed a method for isolating low-abundance proteins using a cysteine-containing glycopeptide strategy. This method involves two orthogonal affinity capture steps. First, glycoproteins are coupled to an azlactone copolymer gel using hydrazide chemistry and cysteine residues are then biotinylated. After trypsinization and extensive washing, tethered N-glycosylated tryptic peptides are released from the gel using PNGase F. Biotinylated cysteinyl-containing glycopeptides are then affinity selected using a monomeric avidin gel and analyzed by LC-MS/MS. We have applied the method to a proteome analysis of mouse plasma. In two independent analyses using 200 muL each of C57BL mouse plasma, 51 proteins were detected. Only 42 proteins were seen when the same plasma sample was analyzed by glycopeptides only. A total of 104 N-glycosylation sites were identified. Of these, 17 sites have hitherto not been annotated in the Swiss-Prot database whereas 48 were considered probable, potential, or by similarity - i.e., based on little or no experimental evidence. We show that analysis by cysteine-containing glycopeptides allows detection of low-abundance proteins such as the epidermal growth factor receptor, the Vitamin K-dependent protein Z, the hepatocyte growth factor activator, and the lymphatic endothelium-specific hyaluronan receptor as these proteins were not detected in the glycopeptide control analysis.
Bernhard
Oliver K
OK
Joint ProteomicS Laboratory, The Ludwig Institute for Cancer Research and the Walter and Eliza Hall Instititute, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia.
Kapp
Eugene A
EA
Simpson
Richard J
RJ
D000818:Animals; D001798:Blood Proteins / Q000032:analysis*; D002853:Chromatography, Liquid; D003545:Cysteine; D006020:Glycopeptides; D006031:Glycosylation; D051379:Mice; D008810:Mice, Inbred C57BL; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014357:Trypsin
D001798:Blood Proteins; D006020:Glycopeptides; D014357:Trypsin; D003545:Cysteine