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Optimizing performance of glycopeptide capture for plasma proteomics.
| Publication Type | Journal Article |
| Authors | Berven, FS, Ahmad R., Clauser KR, and Carr SA |
| Abstract | 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. |
| Year of Publication | 2010 |
| Journal | Journal of proteome research |
| Volume | 9 |
| Issue | 4 |
| Pages | 1706-15 |
| Date Published (YYYY/MM/DD) | 2010/04/05 |
| ISSN Number | 1535-3893 |
| DOI | 10.1021/pr900845m |
| PubMed | http://www.ncbi.nlm.nih.gov/pubmed/20235580?dopt=Abstract |
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