Isobaric protein-level labeling strategy for serum glycoprotein quantification analysis by liquid...

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.