Isobaric
Protein-Level Labeling Strategy for Serum
Glycoprotein Quantification Analysis by Liquid Chromatography–Tandem
Mass Spectrometry
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Abstract
While peptide-level labeling using
isobaric tag reagents has been
widely applied for quantitative proteomics experiments, there are
comparatively few reports of protein-level labeling. Intact protein
labeling could be broadly applied to quantification experiments utilizing
protein-level separations or enrichment schemes. Here, protein-level
isobaric labeling was explored as an alternative strategy to peptide-level
labeling for serum glycoprotein quantification. Labeling and digestion
conditions were optimized by comparing different organic solvents
and enzymes. Digestions with Asp-N and trypsin were found highly complementary;
combining the results enabled quantification of 30% more proteins
than either enzyme alone. Three commercial reagents were compared
for protein-level labeling. Protein identification rates were highest
with iTRAQ 4-plex when compared to TMT 6-plex and iTRAQ 8-plex using
higher-energy collisional dissociation on an Orbitrap Elite mass spectrometer.
The compatibility of isobaric protein-level labeling with lectin-based
glycoprotein enrichment was also investigated. More than 74% of lectin-bound
labeled proteins were known glycoproteins, which was similar to results
from unlabeled and peptide-level labeled serum samples. Finally, protein-level
and peptide-level labeling strategies were compared for serum glycoprotein
quantification. Isobaric protein-level labeling gave comparable identification
levels and quantitative precision to peptide-level labeling