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J Proteome Res DOI:10.1021/pr900845m

Optimizing performance of glycopeptide capture for plasma proteomics.

Publication TypeJournal Article
Year of Publication2010
AuthorsBerven, FS, Ahmad, R, Clauser, KR, Carr, SA
JournalJ Proteome Res
Volume9
Issue4
Pages1706-15
Date Published2010 Apr 05
ISSN1535-3907
KeywordsChromatography, Liquid, Glycopeptides, Glycoproteins, Humans, Hydrazines, Magnetics, Mass Spectrometry, Microspheres, Proteomics, Reproducibility of Results, Sensitivity and Specificity, Sodium Acetate, Sodium Chloride, Solid Phase Extraction
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.

URLhttp://dx.doi.org/10.1021/pr900845m
DOI10.1021/pr900845m
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/20235580?dopt=Abstract

Alternate JournalJ. Proteome Res.
PubMed ID20235580
PubMed Central IDPMC2855884
Grant List1U24 CA126476 / CA / NCI NIH HHS / United States
R01 HL096738 / HL / NHLBI NIH HHS / United States
U24 CA126476-03 / CA / NCI NIH HHS / United States
U24 CA126476 / CA / NCI NIH HHS / United States
R01 HL096738-01 / HL / NHLBI NIH HHS / United States