Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction.

ABSTRACT: Expanding the sequencing depth of the peptides with a statistically significant quantitative change derived from a biological stimulation is critical. Here we demonstrate that optimization of LC gradient and analytical column construction can reveal over 30,000 unique peptides and 23,000 phosphopeptides at high confidence. The quantitative reproducibility of different analytical workflows was evaluated by comparing the phosphoproteome of CD3/4 stimulated and unstimulated T-cells as a model system. A fritless, 50cm-long column packed with 1.9μm particles operated with a standard pressure HPLC significantly improved the sequencing depth 51% and decreased the selected ion chromatogram peak spreading. Most importantly, under the optimal workflow we observed an improvement of over 300% in detec

SUBMITTER: Ahsan N 

PROVIDER: S-EPMC5542054 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature