Optimizing performance of glycopeptide capture for plasma proteomics.
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.
Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Frode S
FS
Berven
Rushdy
R
Ahmad
Karl R
KR
Clauser
Steven A
SA
Carr
D002853:Chromatography, Liquid / Q000379:methods; D006020:Glycopeptides / Q000737:chemistry* / Q000302:isolation & purification; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D008280:Magnetics; D013058:Mass Spectrometry / Q000379:methods; D008863:Microspheres; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019346:Sodium Acetate / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D052616:Solid Phase Extraction / Q000379:methods*
D006020:Glycopeptides; D006023:Glycoproteins; D006834:Hydrazines; C029424:hydrazine; D012965:Sodium Chloride; D019346:Sodium Acetate