iTRAQ-Based Quantitative Protein Expression Profiling and MRM Verification of Markers in Type 2 Diabetes
iTRAQ-Based Quantitative Protein Expression Profiling and MRM Verification of Markers in Type 2 Diabetes
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DOI：
10.1021/pr300798z
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发表时间：
2012-11-01
影响因子：
4.4
通讯作者：
Cheema, Amrita K.
中科院分区：
文献类型：
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作者：
Kaur, Prabhjit;Rizk, Nasser M.;Cheema, Amrita K.
2型糖尿病（T2 DM）的发病机制是复杂的，由于在患病人群中的分子异质性。目前的诊断方法依赖于血糖测量，这对于疾病向其他相关病理的进展是没有信息的。因此，预测T2 DM相关并发症（如心血管疾病）的风险和发展仍然是一个重大挑战。我们使用了一组非糖尿病对照受试者（n = 76）和诊断为T2 DM的患者（n = 106），采用定量方法组合来表征T2 DM循环血清生物标志物。在本病例对照研究中，将样本随机分为训练和验证数据集。在第一步中，进行基于iTRAQ（用于相对和绝对定量的同量异位素标记）的蛋白质表达谱分析，以鉴定在两个研究组中显示显著差异表达的蛋白质。选择这些蛋白质标记物中的五种用于使用多反应监测质谱法（MRM-MS）进行验证，并进一步用Western印迹和QPCR分析进行确认。功能途径分析确定了脂质和小分子代谢的扰动以及导致葡萄糖稳态破坏和血液凝固的途径。这些推定的生物标志物可能在临床上用于T2 DM患者的亚群分层以及针对特定病理的新型治疗剂的开发。
The pathogenesis of Type 2 diabetes mellitus (T2DM) is complex owing to molecular heterogeneity in the afflicted population. Current diagnostic methods rely on blood glucose measurements, which are noninformative with respect to progression of the disease to other associated pathologies. Thus, predicting the risk and development of T2DM-related complications, such as cardiovascular disease, remains a major challenge. We have used a combination of quantitative methods for characterization of circulating serum biomarkers of T2DM using a cohort of nondiabetic control subjects (n = 76) and patients diagnosed with T2DM (n = 106). In this case control study, the samples were randomly divided as training and validation data sets. In the first step, iTRAQ (isobaric tagging for relative and absolute quantification) based protein expression profiling was performed for identification of proteins displaying a significant differential expression in the two study groups. Five of these protein markers were selected for validation using multiple reaction-monitoring mass spectrometry (MRM-MS) and further confirmed with Western blot and QPCR analysis. Functional pathway analysis identified perturbations in lipid and small molecule metabolism as well as pathways that lead to disruption of glucose homeostasis and blood coagulation. These putative biomarkers may be clinically useful for subset stratification of T2DM patients as well as for the development of novel therapeutics targeting the specific pathology.