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.

糖基化（Glycation）在诸多年龄相关性退行性疾病中发挥致病作用，例如糖尿病、终末期肾病与心血管疾病。基于质谱（Mass spectrometry）的定性与定量分析方法已取得长足发展，推动了我们对蛋白质糖基化的认知。然而，在生物样本中灵敏且精准地定量内源性糖基化蛋白质组仍极具挑战。本研究提出一种整合且稳健的定量策略，用于全面解析早期糖基化蛋白质组。该策略采用辅助过滤样品制备法（filter-assisted sample preparation）以减少样品损失、提升样品制备的重现性，从而实现高通量分析与低丰度内源性糖基化蛋白的准确定量。在无标记定量（label-free quantification）与多反应监测（multiple reaction monitoring, MRM）分析中，均将标准糖基化肽段（glycated peptides）掺入复杂样品中同步处理，以此提升定量准确性。与此同时，本研究开发了一种全新方法，用于合成适用于MRM分析的重同位素标记糖基化肽段（heavy isotope-labeled glycated peptides）。通过该策略，从10对伴或不伴心血管并发症的血液透析患者的60次检测中，共鉴定得到1128个内源性糖基化肽段，对应203种血清蛋白；其中234个糖基化肽段（对应63种蛋白）在70%以上的检测中被检出，另有17个肽段呈现显著糖基化差异（P < 0.05，倍数变化>1.5或<0.67）。此外，本研究利用MRM分析验证了46份血清样本中4个目标肽段的糖基化差异，其结果与无标记定量分析结果一致。