Centre for Nano Science and Engineering, Indian Institute of Science, Bengaluru 560012, India.
Samatvam Endocrinology Diabetes Center, Jnana Sanjeevini Diabetes Hospital and Medical Center, Bengaluru, India.
Anal Methods. 2024 Aug 1;16(30):5239-5247. doi: 10.1039/d4ay00503a.
Glycation and the subsequent formation of advanced glycation end products (AGEs) disrupt and impair the physiological functions of proteins. This study presents a comprehensive glycation site mapping of human serum albumin (HSA) utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both glycation experiments and patient samples were investigated, exploring various enzymes, processing techniques, and their impacts on glycation site detection. A pilot study was conducted, analyzing sixteen serum samples, which spanned from healthy individuals to severe diabetic patients (with HbA1c values ranging from 5.7% to 18.1%). The aim was to comprehend the progression of glycation on various sites of HSA with increasing levels of glycation. Their glycated albumin levels (GA) spanned from 19.7% to 62.3%. Trypsin-mediated proteolytic digestion unveiled 12 glycation sites through direct in-solution digestion of whole serum. However, isolating albumin from serum enabled the identification of a higher number of glycation sites in each sample compared to direct serum digestion. Boronate affinity chromatography facilitated the segregation of less glycated albumin (LGA) from the more glycated albumin (MGA) fraction. Subsequent proteolytic digestion of both LGA and MGA samples revealed similar glycation sites. The MGA fraction exhibited a greater number of identified glycation sites, thereby elucidating which sites are particularly prone to glycation in highly glycated albumin samples. Changes in relative glycation levels were noted in the tryptic digests of albumin samples following the sample enrichment steps, as opposed to direct in-solution digestion of whole serum. Two enzymes, trypsin and Glu-C, were evaluated for efficacy in sequence coverage and glycation site analysis of HSA, with trypsin demonstrating superior efficiency over Glu-C.
糖基化作用和随后形成的晚期糖基化终产物 (AGEs) 会破坏和损害蛋白质的生理功能。本研究利用液相色谱-串联质谱 (LC-MS/MS) 对人血清白蛋白 (HSA) 进行了全面的糖基化位点映射。研究了糖化实验和患者样本，探索了各种酶、处理技术及其对糖化位点检测的影响。进行了一项试点研究，分析了 16 个血清样本，范围从健康个体到严重糖尿病患者 (HbA1c 值从 5.7%到 18.1%)。目的是了解随着糖化程度的增加，HSA 各个部位糖化的进展情况。他们的糖化白蛋白水平 (GA) 从 19.7%到 62.3%不等。胰蛋白酶介导的蛋白水解消化通过直接在溶液中消化整个血清揭示了 12 个糖基化位点。然而，与直接血清消化相比，从血清中分离白蛋白可以在每个样本中鉴定出更多的糖基化位点。硼酸盐亲和色谱有助于将较少糖化的白蛋白 (LGA) 与更多糖化的白蛋白 (MGA) 部分分离。随后对 LGA 和 MGA 样本进行蛋白水解消化揭示了相似的糖基化位点。MGA 部分鉴定出的糖基化位点数量更多，从而阐明了在高度糖化的白蛋白样本中哪些位点特别容易发生糖化。与直接在溶液中消化整个血清相比，在样品富集步骤后，白蛋白样品中胰蛋白酶消化的相对糖基化水平发生了变化。评估了两种酶，胰蛋白酶和 Glu-C，用于 HSA 的序列覆盖率和糖基化位点分析，胰蛋白酶的效率优于 Glu-C。
