Journal
JOURNAL OF PROTEOME RESEARCH
Volume 9, Issue 4, Pages 1706-1715Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr900845m
Keywords
solid phase extraction of N-linked glycoproteins; proteomics; glycoproteins; hydrazide chemistry; biomarker discovery; glycopeptide
Categories
Funding
- Bill and Melinda Gates Foundation
- National Institutes of Health [1U24 CA126476]
- NCI's Clinical Proteomic Technologies Assessment in Cancer Program
- National Heart Lung and Blood Institute [R01 HL096738-01]
- Women's Cancer Research Fund of the Entertainment Industry Foundation
- Western Norway Regional Health Authority
- Norwegian Research Council
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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.
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