A HUPO TEST SAMPLE STUDY REVEALS COMMON PROBLEMS IN MASS SPECTROMETRY-BASED PROT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们进行了一项测试样本研究，试图在基于液相色谱-质谱仪的蛋白质组学中识别导致不可重复性的错误，包括肽采样的不完整性。我们向27个实验室分发了一个等摩尔测试样本，其中包含20个高度纯化的重组人类蛋白。每种蛋白都含有一个或多个1250Da的独特胰蛋白酶多肽，用于质谱仪中的离子选择和采样。在27个实验室中，只有7个实验室的成员最初报告的所有20种蛋白质都是正确的，只有1个实验室的成员报告了所有1250Da的胰蛋白酶多肽。然而，对原始数据的集中分析显示，在所有27个实验室中都检测到了所有20种蛋白质和大部分1,250大肽。我们的集中分析将遗漏的鉴定(假阴性)、环境污染、数据库匹配和蛋白质鉴定的精选确定为问题的根源。基于质谱学的蛋白质组学需要改进的搜索引擎和数据库。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography-mass spectrometry-based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry-based proteomics.