Enhanced Analysis of the Mouse Plasma Proteome Using Cysteine-Containing Tryptic Glycopeptides
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- 作者：Oliver K. Bernhard ; Eugene A. Kapp ; Richard J. Simpson
- 刊名：Journal of Proteome Research
- 出版年：2007
- 出版时间：March 2007
- 年：2007
- 卷：6
- 期：3
- 页码：987 - 995
- 全文大小：208K
- 年卷期：v.6,no.3(March 2007)
- ISSN：1535-3907
文摘
A comprehensive understanding of the mouse plasma proteome is important for studies using mousemodels to identify protein markers of human disease. To enhance our analysis of the mouse plasmaproteome, 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 cysteineresidues are then biotinylated. After trypsinization and extensive washing, tethered N-glycosylated trypticpeptides are released from the gel using PNGase F. Biotinylated cysteinyl-containing glycopeptidesare then affinity selected using a monomeric avidin gel and analyzed by LC-MS/MS. We have appliedthe method to a proteome analysis of mouse plasma. In two independent analyses using 200 
L eachof C57BL mouse plasma, 51 proteins were detected. Only 42 proteins were seen when the same plasmasample was analyzed by glycopeptides only. A total of 104 N-glycosylation sites were identified. Ofthese, 17 sites have hitherto not been annotated in the Swiss-Prot database whereas 48 were consideredprobable, potential, or by similarity - i.e., based on little or no experimental evidence. We show thatanalysis by cysteine-containing glycopeptides allows detection of low-abundance proteins such as theepidermal growth factor receptor, the Vitamin K-dependent protein Z, the hepatocyte growth factoractivator, and the lymphatic endothelium-specific hyaluronan receptor as these proteins were notdetected in the glycopeptide control analysis.
L eachof C57BL mouse plasma, 51 proteins were detected. Only 42 proteins were seen when the same plasmasample was analyzed by glycopeptides only. A total of 104 N-glycosylation sites were identified. Ofthese, 17 sites have hitherto not been annotated in the Swiss-Prot database whereas 48 were consideredprobable, potential, or by similarity - i.e., based on little or no experimental evidence. We show thatanalysis by cysteine-containing glycopeptides allows detection of low-abundance proteins such as theepidermal growth factor receptor, the Vitamin K-dependent protein Z, the hepatocyte growth factoractivator, and the lymphatic endothelium-specific hyaluronan receptor as these proteins were notdetected in the glycopeptide control analysis.