纳升液相色谱-质谱分析方法的重现性对尿液多肽组分析结果的影响

多肽组学是蛋白质组学技术的延伸和扩展,在医学和生物学研究中的应用日益广泛,但是,多肽组鉴定方法的重现性对实验结果的影响目前尚不清楚.本研究利用纳升液相色谱-高分辨质谱对健康人的尿液多肽组进行了7次平行分析,考察图谱数目、图谱利用率、鉴定的肽段数目、蛋白质数目、样品总离子强度和肽段保留时间等指标的变化,以揭示重复实验之间分析结果的可变性和稳定性.7次测定的肽段数目平均值为208,标准偏差为38;7次结果合并后,得到了归属于114个蛋白质的426个肽段,肽段和蛋白质数目均显著增加;而35个蛋白质的109个肽段在所有7次实验中均被检出,表明多肽组的单次分析结果既具有一定的随机性,又具有相对的稳定性.增加平行实验次数会扩大多肽组数据集,但测定3次以上后增加幅度减小.相比于肽段,多肽组的结果在蛋白质水平上更为稳定,提示利用蛋白质为多肽组的生物标志物更为稳健.

Effect of Reproducibility of Nano-liquid Chromatography-Mass Spectrometry on Analysis of Urinary Peptidomics

As an extension of proteomics, peptidomics has been widely used in medical and biological researches. However, the effect of reproducibility of identification method on peptidomics is not yet clear. In this work, the urine sample of a healthy people was analyzed for seven times in parallel by nano-liquid chromatography-high-resolution tandem mass spectrometry. To illustrate the variability and stability among these experiments, the number of spectra, the utilization of total spectra, the number of identified peptides, the number of proteins, and the ionic strength and retention time of peptides were counted and compared. The average number of peptides was 208 and the standard deviation was 38. 7. After all of data were combined, 426 peptides belonging to 114 proteins were obtained, while only 109 peptides coming from 35 proteins were identified in each experiment, indicating that there were both an randomness and a relative stability for LC-MS analysis. Increasing the number of parallel experiments would expand the data set of peptidomics, but the rate of increase would decrease over 3 or more measurements. Compared with peptides, the results of peptidomics were more stable at protein level, indicating that proteins were more robustly peptidomics biomarker than the peptides.