人血清内源性多肽无标记定量分析新策略

A strategy with label-free quantification of the targeted peptides for quantitative peptidome analysis of human serum

多肽组学(Pepti domics)以其在疾病标志物发现中所起的重要作用正受到越来越多的关注.然而,目前尚缺少针对多肽组学的高效定量方法.本文建立了一种基于纳升液相色谱与四极杆飞行时间质谱联用(μLC-Q-TOF-MS/MS)系统的色谱峰面积无标记多肽定量方法.该方法首先对两组样品中的多肽进行相对定量,然后选取相对量大于2或小于0.5的多肽,用纳升液相色谱与串联二级质谱(μLC-MS/MS)的选择性数据依赖模式(DDA)进行序列鉴定.将不同量标准样品牛血清白蛋白(BSA)酶解液加入到相同量的正常人血清样品中,然后用该方法进行定量分析,得到的定量结果平均相对偏差为6.42%.该方法分别被用于健康人血清与肝细胞癌(HCC)以及乳腺癌病人血清中内源性多肽的定量比较分析,并成功鉴定了血清中一些与HCC和乳腺癌相关的内源性多肽.由于Q-TOF-MS/MS的高分辨率和宽质量检测范围,价态高达+5和分子量超过4000的多肽也可被可靠地进行定性和定量分析.实验结果进一步表明在多肽组学分析中μLC-Q-TOF-MS/MS比传统的MALDI-TOF-MS具有更高的检测灵敏度.

Peptidomics draws more and more attention in discovering useful biomarkers for early diagnosis of disease.However,there is lack of efficient quantification strategy in peptidome analysis.In this study,a strategy with label-free quantification of the targeted endogenous peptides based on peak intensity using μUPLC-Q-TOF-MS/MS was developed for quantitative peptidome analysis of human serum.Different amounts of standard BSA tryptic digesting peptides were added into the same serum extracts for evaluation of the developed strategy,and it was observed that the average relative error of the targeted peptides was 6.42%,which was superior to the result obtained directly by commercially available software PLGS.It was also demonstrated this quantification strategy could obviously increase the detection sensitivity of the peptide by DDA analysis.Then,this strategy was applied to comparatively analyze the peptides extracted from the serum of HCC or breast cancer patients and healthy individuals,respectively.Peptides with charge states up to 5 and molecular weight over 4000 can be reliably identified and quantified.And this quantitative analysis method based on μUPLC-Q-TOF-MS/MS exhibited superior sensitivity than that by MALDI-TOF-MS commonly used in peptidome analysis.Finally,some interesting endogenous peptides related to corresponding diseases were successfully obtained.