In proteome analysis, HeLa cell protein digest is used as proteome standard for quality control of LC-MS/MS system and optimization of experimental parameters. When a large number of samples are analyzed, the standard sample needs to be analyzed periodically to check the performance of LC-MS/MS. During this process, the standard can be exposed to air for a long time in auto-sampler, it causes peptide degradation by air oxidation and acidic buffer condition. Such degradation affects proteome analysis based on peptide identification, and consequently reduces the reliability of the experiment. ^[1] Herein, we examined which peptides were degraded under air expose and acidic storage condition from HeLa cell protein digest during the experimental period of about two weeks. HeLa cell protein digests diluted with acidic buffer (water containing 0.1% formic acid) were stored for 1 to 16 days at room temperature and 4 ° C. Those air-exposed samples were analyzed by LC-MS/MS followed identification using IP2(Integrated Proteomics Pipeline). In the comparisons with fresh sample, HeLa cell protein digest samples exposed to air for more than 4 days at room temperature gave fewer peptides and proteins identification. Easily degraded peptides were identified and oxidation patterns at methionine or cysteine were investigated. These peptides can be used to determine the degree of degradation of HeLa cell protein digest. This study will be helpful in testing the performance of LC-MS/MS system required for long term large-scale proteome analysis.

[1] Sen, K. I., Hepler, R., & Nanda, H. (2017). Detection and Measurement of Methionine Oxidation in Proteins. Current Protocols in Protein Science, 87(1), 14-16.