糖尿病视网膜病变血浆差异蛋白分析

Plasma proteomics research in patients with diabetic retinopathy

目的利用蛋白质组学技术寻找糖尿病视网膜病变(DR)患者血浆中的差异蛋白,为筛选DR相关血浆标志物提供依据。方法选取在解放军第903医院诊治的2型糖尿病(T2DM)患者14例,其中合并中度以上非增殖性DR(NPDR)或增殖性DR(PDR)者作为DR组(n=8),不合并DR者作为NDR组(n=6)。对患者的血浆标本利用蛋白质非标记(label-free)定量技术行液相串联质谱分析(LC-MS/MS),检测血浆中各个蛋白的表达丰度,筛选两组间的差异蛋白,对其进行生物信息学分析,包括基因本体(GO)和代谢通路(Pathway)注释,GO和Pathway富集分析、差异蛋白相互作用网络分析。结果以均数和中位数差异倍数≥2.5倍为标准,共筛选出DR与NDR患者血浆中的差异蛋白41个,其中上调者26个,下调者15个。GO分析显示,差异蛋白中结合功能蛋白占绝对优势,生物过程中单一组织过程占比最高,细胞组分中细胞和细胞部分占比最高。Pathway富集性分析显示,上调最显著的原肌球蛋白4(TPM4)与肌肉收缩调节有关,下调最显著的血小板膜蛋白V(GPV)在细胞外基质受体相互作用中起重要作用。蛋白相互作用网络分析显示,3-磷酸甘油醛脱氢酶(GAPDH)与巯基氧化酶1(QSOX1)、免疫球蛋白kappa基因座(IGK@)、TPM4、载脂蛋白C2(APOC2)、免疫球蛋白重链可变区4-31(IGHV4-31)之间可能有相互作用;妊娠区带蛋白质(PZP)与金属蛋白酶组织抑制剂2(TIMP2)之间可能有相互作用。结论采用蛋白质非标记定量技术发现DR与NDR患者血浆存在多种差异表达蛋白,这些差异蛋白提供了DR候选血浆标志物,并可能成为DR早期防治的候选靶点。

Objective To screen potential DR-related plasma markers by profiling the plasma proteomics in patients with diabetic retinopathy(DR). Methods A total of 8 patients with moderate or higher non-proliferative diabetic retinopathy(NPDR)or proliferative diabetic retinopathy(PDR) enrolled in our hospital are included in the DR group and 6 patients without DR as NDR group. Plasma samples from these 14 patients were subjected to protein-labeled-free quantification and parallel liquid-phase tandem mass spectrometry(LC-MS/MS) in order to calculate the relative protein abundance of each protein detected in plasma. The results were statistically analyzed to find significant differences between the two groups of proteins. Further, bioinformatics analysis was performed, including gene ontology(GO) and Pathway annotation, GO and Pathway enrichment analysis, and protein interaction network analysis. Results A total of 41 differential proteins were identified with mean and median ratios ≥2.5(up/down). Among them, 26 were up-regulated and 15 were down-regulated. GO analysis showed that the binding protein in the differential protein was dominant, the single-organism process in the biological process accounted for the highest proportion, and the cell and cell part in the cell component accounted for the highest proportion. Pathway enrichment analysis indicated that the most significant upregulated protein, tropomyosin 4(TPM4), is associated with regulation of muscle contraction;while the most prominent downregulated protein, platelet membrane glycoproteins V(GPV), plays an important role in extracellular matrix(ECM) receptor interactions. Protein interaction network analysis showed that there were potential interactions among glyceraldehyde-3-phosphate dehydrogenase(GAPDH), sulfhydryl oxidase 1(QSOX1), immunoglobulin kappa locus(IGK@), TPM4, apolipoproteins C2(APOC2), and immunoglobulin heavy variable 4-31(IGHV4-31);another potential interaction between pregnancy zone protein(PZP) and tissue inhibitor of metalloproteinase 2(TIMP2). Conclusion Profiling the proteomics of DR vs NDR with label-free quantification technology successfully identified differentially expressed proteins in DR and NDR. These differential proteins are potential DR associated plasma markers and maybe a new target for early prevention and treatment of DR.