Shenzhen Second People's Hospital , The First Affiliated Hospital of Shenzhen University , Shenzhen 518020 , China.
Integrated Chinese and Western Medicine Postdoctoral Research Station , Jinan University , Guangzhou 510632 , China.
Anal Chem. 2020 Feb 18;92(4):3237-3245. doi: 10.1021/acs.analchem.9b05046. Epub 2020 Jan 31.
Proteins in a human body continuously undergo glycation reactions with reducing sugars, forming early as well as advanced glycation end-products (AGEs) which are highly disease-relevant. Specifically, -1-(deoxyfructosyl) valine of β-hemoglobin (HbA1c) has been considered as a marker of diabetes, but the exact map of glycated Hb peptides corelated with diabetes in different stages is poorly studied. Here, the pseudotargeted parallel reaction monitoring (PRM) method combined with relative retention time (iRT) endogenous peptides was proposed for exploring the roles of deoxyfructosyl (DF-), carboxymethyl (CM-), and carboxyethyl (CE-) based Hb modifications in clinical prognosis and diagnosis of type 2 diabetes mellitus (T2DM) and its complication. For building the pseudotargeted list, data-dependent acquisition (DDA) combined with multiple enzyme digestion was employed for the comprehensive identification of the three types of modification in vitro Hb and in vivo Hb. The introduction of the endogenous iRT peptides during PRM analysis facilitates being able to obtain accurate quantitative results. When applying this new strategy to quantify the three kinds of glycated Hb peptides in clinical samples, patients with T2DM in different pathophysiological conditions were fully distinguished from the controls, indicating the necessity of adopting multiple glycation types for the improved diagnosis of T2DM. Taken together, the newly developed pseudotargeted PRM method not only expands the horizons of glycated Hb by reliably assessing the actual status of T2DM but also reveals that endogenous iRT might be a viable option for label-free quantitative analysis.
人体内的蛋白质不断与还原糖发生糖化反应，形成早期和晚期糖化终产物（AGEs），这些产物与疾病高度相关。具体来说，β-血红蛋白（HbA1c）中的-1-（脱氧果糖基）缬氨酸被认为是糖尿病的标志物，但不同阶段糖尿病相关糖化 Hb 肽的精确图谱研究甚少。在这里，提出了一种伪靶向平行反应监测（PRM）方法结合相对保留时间（iRT）内源性肽，用于探索基于脱氧果糖基（DF-）、羧甲基（CM-）和羧乙基（CE-）的 Hb 修饰在 2 型糖尿病（T2DM）及其并发症的临床预后和诊断中的作用。为了构建伪靶向列表，采用数据依赖采集（DDA）结合多种酶消化的方法，对三种修饰类型的体外 Hb 和体内 Hb 进行了全面鉴定。在 PRM 分析中引入内源性 iRT 肽有助于获得准确的定量结果。当将这种新策略应用于定量分析临床样本中的三种糖化 Hb 肽时，不同病理生理条件下的 T2DM 患者与对照组完全区分开来，表明需要采用多种糖化类型来提高 T2DM 的诊断能力。总之，新开发的伪靶向 PRM 方法不仅通过可靠地评估 T2DM 的实际状况扩展了糖化 Hb 的研究范围，还表明内源性 iRT 可能是一种可行的无标记定量分析选择。
