Online 2D-LC-MS/MS Platform for Analysis of Glycated Proteome

Glycated proteins are emerging as
good indicators for diabetes
and age related diseases. However, the platform for analysis of glycated
proteome has been relatively less well established. We here introduce
an online 2D-LC-HCD-MS/MS platform for comprehensive glycated peptide
quantification. This platform includes a boronate affinity column
in the first dimension for enrichment, reversed phase nanoLC column
in the second dimension for separation, a benchtop Orbitrap mass spectrometer
with HCD-MS/MS for peptide sequencing, and MaxQuant bioinformatics
tool for identification and quantification of glycated peptides. This
online 2D-LC-HCD-MS/MS platform has high enrichment efficiency with
85% of identified peptides in the enriched fraction as glycated, high
sensitivity for detection of glycated peptides with LOD and LOQ at
1.2 and 2.4 pg, respectively, and high reproducibility with interday
CVs < 20% for 80% of the glycated peptides. The number of glycated
peptides quantified in in vitro glycated human plasma
increased more than 3-fold using this platform in comparison to that
obtained using 1D-LC-HCD-MS/MS platform without boronate affinity
enrichment. Application of this online platform to human plasma identified
376 glycated peptides from 10 μg of protein digests. This highly
sensitive and reproducible online 2D platform is promising for glycated
protein analysis of complex clinical samples.