心血管并发症血液透析患者糖化候选生物标记物发现和验证的综合策略,Analytical Chemistry

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心血管并发症血液透析患者糖化候选生物标记物发现和验证的综合策略
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-03-04 , DOI: 10.1021/acs.analchem.0c04028
Linlin Wu ₁ , Caiyun Fang ₁ , Lei Zhang ₂ , Wenjuan Yuan ₂ , Xiaofang Yu ₃ , Haojie Lu _{1,

2}

Affiliation

糖基化在许多与年龄有关的退化性病理状况中发挥致病作用，例如糖尿病，终末期肾脏疾病和心血管疾病。基于质谱的定性和定量分析方法已得到很大发展，并有助于我们对蛋白质糖基化的理解。然而，灵敏而准确地定量生物样品中的内源糖基化蛋白质组仍然具有挑战性。在本文中，我们提出了一种综合而强大的定量策略，用于对早期糖基化蛋白质组进行综合分析。在这种策略中，采用了过滤辅助样品制备方法来减少样品损失并提高样品制备的可重复性，从而有助于高通量分析和低丰度内源糖基化蛋白质的准确定量。掺入标准糖基化肽，并在无标记定量和多反应监测（MRM）分析中与复杂样品一起执行后续过程，从而有助于提高定量准确性。同时，开发了一种新颖的方法来合成用于MRM分析的重同位素标记的糖基化肽。通过这种方法，从10对有和无心血管并发症的血液透析患者的60例中鉴定出总共1128种内源糖基化肽，对应于203种血清蛋白，其中> 63％的患者中存在234种对应于63种蛋白的糖基化肽，其中发现17种肽的糖基化程度不同（有助于提高定量精度。同时，开发了一种新颖的方法来合成用于MRM分析的重同位素标记的糖基化肽。通过这种方法，从10对有和无心血管并发症的血液透析患者的60个运行中鉴定出总共1128个内源糖基化肽，对应于203个血清蛋白，其中> 63％的血液中存在234个对应于63个蛋白的糖基化肽，其中发现17种肽的糖基化程度不同（有助于提高定量精度。同时，开发了一种新颖的方法来合成用于MRM分析的重同位素标记的糖基化肽。通过这种方法，从10对有和无心血管并发症的血液透析患者的60个运行中鉴定出总共1128个内源糖基化肽，对应于203个血清蛋白，其中> 63％的血液中存在234个对应于63个蛋白的糖基化肽，其中发现17种肽的糖基化程度不同（P <0.05，倍数变化> 1.5或<0.67）。此外，我们使用MRM分析验证了46种血清样品中四种目标肽的糖基化差异，这与我们的无标记定量分析结果一致。

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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.

更新日期：2021-03-16

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