Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS

J Proteome Res. 2016 Dec 2;15(12):4146-4164. doi: 10.1021/acs.jproteome.5b01159. Epub 2016 Nov 9.


Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC-MS/MS platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow. 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was <25% for all test samples. Our analytical protocol yielded identification and quantification of site-specific N-glycopeptide isoforms of AGP from control and disease plasma sample.

Keywords: PTM (post translational modification); interlaboratory study; isoforms; site-specific N-glycopeptide.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Blood Specimen Collection
  • Chromatography, Liquid
  • Glycopeptides / chemistry*
  • Glycosylation
  • Humans
  • Orosomucoid / chemistry*
  • Protein Isoforms / analysis*
  • Reproducibility of Results
  • Tandem Mass Spectrometry


  • Glycopeptides
  • Orosomucoid
  • Protein Isoforms