Integrated Strategy for Discovery and Validation of Glycated Candidate Biomarkers for Hemodialysis Patients with Cardiovascular Complications

Glycation
plays a pathogenic role in many age-related degenerative
pathological conditions, such as diabetes, end-stage renal diseases,
and cardiovascular diseases. Mass spectrometry-based qualitative and
quantitative analysis methods have been greatly developed and contribute
to our understanding of protein glycation. However, it is still challenging
to sensitively and accurately quantify endogenous glycated proteome
in biological samples. Herein, we proposed an integrated and robust
quantitative strategy for comprehensive profiling of early-stage glycated
proteome. In this strategy, a filter-assisted sample preparation method
was applied to reduce sample loss and improve reproducibility of sample
preparation, contributing to high-throughput analysis and accurate
quantification of endogenous glycated proteins with low abundance.
Standard glycated peptides were spiked and performed the subsequent
process together with complex samples both in label-free quantification
and multiple reaction monitoring (MRM) analysis, contributing to the
improvement of quantitative accuracy. In parallel, a novel approach
was developed for the synthesis of heavy isotope-labeled glycated
peptides used in MRM analysis. By this way, a total of 1128 endogenous
glycated peptides corresponding to 203 serum proteins were identified
from 60 runs of 10 pairs of hemodialysis patients with and without
cardiovascular complications, and 234 glycated peptides corresponding
to 63 proteins existed in >70% runs, among which 17 peptides were
discovered to be differentially glycated (P <
0.05, fold-change > 1.5 or <0.67). Furthermore, we validated
the
glycation difference of four target peptides in 46 serum samples using
MRM analysis, which were consistent with our results of label-free
quantification.