Optimization of pretreatment methods for ubiquitination proteomics of micro-samples
Objective:To enhance the ability of tandem mass spectrometry based-proteomics methods for detecting ubiquitina-tion modification sites in trace samples and increase the coverage of ubiquitination modification identification.This paper systemati-cally evaluates the factors affecting the enrichment of ubiquitination peptides,hoping to provide a reference method for the study of ubiquitination modification proteomics in trace clinical samples.Methods:In this study,we used 100 μg HeLa protein digest pep-tides to systematically evaluate the effects of different conditions on enrichment and identification ability of ubiquitinated peptides and sites.During ubiquitination enrichment,the ability to identify ubiquitinated peptides and sites was investigated at different anti-bodies dosages(9.375 μg,15.625 μg,and 31.25 μg)and enrichment volumes(300 μL,600 μL,and 1 000 μL),and the effect of adding dimethyl sulfoxide before lyophilization on the improvement of ubiquitinated peptide identification ability was also investigat-ed.Results:The enrichment and identification efficiency of ubiquitination peptides varied with different enrichment conditions.By evaluating the amount of antibody input,15.625 μg bead-conjugated antibodies result in the best enrichment efficiency,up to 57%,and better identification quantities in both ubiquitinated peptides and sites.By comparing the effects of enrichment volumes,600 μL enrichment buffer resulted in more than 10 000 ubiquitination peptides and sites in a single injection,and the quantitative correlation coefficient of the ubiquitinated sites in the two replicate experiments reached 0.95.The addition of dimethyl sulfoxide before lyophilization improved the identification capacity of hydrophobic peptides,resulting in an increase of 4.59%in the identi-fied ubiquitination sites.Conclusion:After systematic optimization,15.625 μg of bead-conjugated antibodies and 600 μL system volumes were recommended for the enrichment of 100 μg peptides,and the addition of dimethyl sulfoxide before lyophilization could effectively increase the identification of hydrophobic peptides.
万方数据
维普
