Biomedical Proteomics Research Group, Department of Structural Biology and Bioinformatics, University Medical Centre, University of Geneva, 1211 Geneva 4, Switzerland.
Mol Cell Proteomics. 2010 Mar;9(3):579-92. doi: 10.1074/mcp.M900439-MCP200. Epub 2009 Nov 6.
Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [(13)C(6)]glucose incubation to evaluate the native glycated proteins labeled with [(12)C(6)]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration.
蛋白质的非酶糖基化是一种翻译后修饰，由还原糖与赖氨酸和精氨酸残基或 N 末端位置的氨基基团之间的反应产生。这种修饰在医学和食品工业中起着重要作用。在临床领域，这种不期望的作用与血糖浓度直接相关，因此与高血糖（>11 毫米葡萄糖）引起的病理状况直接相关，如糖尿病或肾衰竭。本文提出了一种定性和定量分析糖化蛋白的方法，以实现其完全特征化的三个信息水平。这些是：1）鉴定糖化蛋白，2）阐明糖基化位点，3）定量分析以比较血糖状态。定性分析是通过内切蛋白酶 Glu-C 消化和硼酸盐亲和色谱分离糖化肽后进行串联质谱分析来完成的。为此，使用了两种 MS 操作模式：更高能量碰撞解离-MS2 和 CID-MS3 通过监测两种选择性中性丢失（162.05 和 84.04 Da 用于葡萄糖裂解和葡萄糖部分的中间重排）的中性丢失扫描进行分析。另一方面，定量分析基于用 [(13)C(6)]葡萄糖孵育标记蛋白质，以评估用 [(12)C(6)]葡萄糖标记的天然糖化蛋白质。由于糖化是化学选择性的，因此仅发生在体内修饰的潜在靶标中。这种方法称为糖化同位素标记，通过质谱（6-Da 质量位移/糖化位点）对由酶消化产生的两种同位素形式标记的糖化肽进行区分。然后将该策略应用于参考血浆样品，检测到 50 种糖化蛋白和 161 个糖基化位点，并鉴定了每种蛋白质的优先糖化位点。还测试了一种预测方法，以在高葡萄糖浓度下检测潜在的糖化位点。
