Optimizing performance of glycopeptide capture for plasma proteomics

J Proteome Res. 2010 Apr 5;9(4):1706-15. doi: 10.1021/pr900845m.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Liquid / methods
  • Glycopeptides / chemistry*
  • Glycopeptides / isolation & purification
  • Glycoproteins / chemistry
  • Glycoproteins / isolation & purification
  • Humans
  • Hydrazines / chemistry
  • Magnetics
  • Mass Spectrometry / methods
  • Microspheres*
  • Proteomics / methods*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sodium Acetate / chemistry
  • Sodium Chloride / chemistry
  • Solid Phase Extraction / methods*

Substances

  • Glycopeptides
  • Glycoproteins
  • Hydrazines
  • hydrazine
  • Sodium Chloride
  • Sodium Acetate