iProXProteomicsPiliang HaoHomo SapiensEscherichia Colihttp://www.iprox.org/page/project.html?id=IPX0001174000haopl@shanghaitech.edu.cnShanghaiTech UniversityiProXHigh specificity of trypsin is a prerequisite for accurate identification and quantification of proteins in shotgun proteomics. It is important to minimize nonspecific enzymatic cleavages during proteomic sample preparation.<h4>Methods</h4>In this study, protein extraction and trypsin digestion conditions were extensively evaluated using the less-complex Escherichia coli lysates to improve the sensitivity of detecting low-abundance nonspecific peptides by liquid chromatography/tandem mass spectrometry.<h4>Results</h4>Trypsin digestion buffers and digestion times were proved to have a significant effect on nonspecific cleavages. The triethylammonium bicarbonate buffer induces significantly lower nonspecific cleavages than the other two buffers, but a freshly prepared urea solution does not induce more than sodium dodecyl sulfate. Because prolonged trypsin digestion resulted in a considerable number of nonspecific cleavages, an optimized 2-h protocol was developed with 45.2% less semispecific tryptic peptides but 18.5% more unmodified peptides identified than the commonly used 16-h protocol.<h4>Conclusions</h4>The significant decrease in nonspecific cleavages and artificial modifications improves the accuracy of protein quantification and the identification of low-abundance proteins, and it is especially useful for studying protein posttranslational modifications. For trypsin digestion, the proposed 2-h protocol can potentially be a replacement for the traditional 16-h protocol.Evaluation and minimization of nonspecific tryptic cleavages in proteomic sample preparation.Lin Zhilong Z, Ren Yan Y, Shi Zhaomei Z, Zhang Keren K, Yang Huanming H, Liu Siqi S, Hao Piliang PfalseEvaluation and minimization of nonspecific tryptic cleavages in proteomic sample preparationDataset for evaluation of nonspecific cleavages and artificial modifications using E.coli and A549 cellsSun Jan 19 00:00:00 GMT 2020PXD017186562960632031715