Optimizing Performance of Glycopeptide Capture for Plasma Proteomics

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• 作者：Frode S. Berven ; Rushdy Ahmad ; Karl R. Clauser ; Steven A. Carr
• 刊名：Journal of Proteome Research
• 出版年：2010
• 出版时间：April 5, 2010
• 年：2010
• 卷：9
• 期：4
• 页码：1706-1715
• 全文大小：215K
• 年卷期：v.9,no.4(April 5, 2010)
• ISSN：1535-3907

文摘

Selective capture of glycopolypeptides followed by release and analysis of the former glycosylation-site peptides has been shown to have promise for reducing the complexity of body fluids such as blood for biomarker discovery. In this work, a protocol based on capture of polypeptides containing a N-linked carbohydrate from human plasma using commercially available magnetic beads coupled with hydrazide chemistry was optimized and partially automated through the use of a KingFisher magnetic particle processor. Comparison of bead-based glycocapture at the protein-level vs the peptide-level revealed differences in the specificity, reproducibility, and absolute number of former glycosylation-site peptides detected. Evaluation of a range of capture and elution conditions led to an optimized protocol with a 24% intraday and 30% interday CV and a glycopeptide capture specificity of 99%. Depleting the plasma of 14 high abundance proteins improved detection sensitivity by approximately 1 order of magnitude compared to nondepleted plasma and resulted in an increase of 24% in the number of identified glycoproteins. The sensitivity of SPEG for detection of glycoproteins in depleted, non-fractionated plasma was found to be in the 10−100 pmol/mL range corresponding to glycoprotein levels ranging from 100's of nanograms/mL to 10's of micrograms/mL. Despite high capture specificity, the total number of glycoproteins detected and the sensitivity of SPEG in plasma is surprisingly limited.