Department of Chemistry , Fudan University , Shanghai 200433 , People's Republic of China.
Anal Chem. 2018 Mar 20;90(6):3752-3758. doi: 10.1021/acs.analchem.7b03668. Epub 2018 Mar 1.
Due to the critical role glycation plays in many serious pathological conditions, such as diabetes, it is of great significance to discover protein glycation at an early stage for precaution and prediction of the disease. Here, a method of reductive amination combining dimethylation (RAD) was developed for the quantification of early-stage glycated proteins. The quantitative analysis was first carried out by reducing the samples using NaBHCN or NaBDCN, resulting in a 1 Da mass shift and the stabilization of early-stage protein glycation. The two samples were then digested and isotopically dimethylated to achieve the mass shift of 4 m + 3 n ( m represents the number of N-termini and Lys residues, and n represents the number of glycated sites) between light- and heavy-labeled glycated peptides for quantification. Consequently, the false positive result can be removed according to the different mass shifts of glycated peptides and non-glycated peptides. In quantification of glycated myoglobin, RAD showed good linearity ( R > 0.99) and reproducibility (CVs ≤ 1.6%) in 2 orders of magnitude (1:10-10:1). RAD was then applied to quantify the endogenous glycated proteins in the serum of diabetic patients, revealing significant differences in the glycation level between the patients with complicated retinal detachment and those without. In conclusion, RAD is an effective method for quantifying endogenous glycated proteins.
由于糖基化在许多严重的病理条件中（如糖尿病）中起着关键作用，因此尽早发现蛋白质糖基化对于疾病的预防和预测具有重要意义。在这里，我们开发了一种结合了二甲基化（RAD）的还原胺化方法，用于定量早期糖基化蛋白。首先通过使用 NaBHCN 或 NaBDCN 将样品还原，从而导致 1 Da 的质量位移和早期蛋白质糖基化的稳定，从而进行定量分析。然后，将两个样品进行消化并进行同位素二甲基化，以实现轻标记和重标记糖基化肽之间 4 m + 3 n（m 代表 N-末端和赖氨酸残基的数量，n 代表糖基化位点的数量）的质量位移，从而进行定量。因此，可以根据糖基化肽和非糖基化肽的不同质量位移来去除假阳性结果。在糖化肌红蛋白的定量中，RAD 在 2 个数量级（1:10-10:1）内表现出良好的线性（R > 0.99）和重现性（CVs ≤ 1.6%）。然后，RAD 被应用于定量糖尿病患者血清中的内源性糖基化蛋白，结果表明，伴有视网膜脱离并发症的患者与无并发症的患者之间的糖化水平存在显著差异。总之，RAD 是一种有效的定量内源性糖基化蛋白的方法。
