Label-free quantitation to dissect site-specific phosphorylation stoichiometry and dynamics of signaling proteins involved in inflammation and pathogenesis

摘要

Label-free quantitative proteomics is the method of choice for comparative investigations of large sample sets. In principal, infinite numbers of data points are captured in a time-course study without the need for in vivo labeling or chemical tagging. However, label-free quantitation usually requires multiple technical/biological replicates and software tools capable of alignment of data for quantitative analysis. However, these tools have limited flexibility to target site-specific phosphorylation analysis in large-scale discovery proteomics. In particular, the non-uniform sampling of peptides due to missed cleavages, and artifactual modifications can affect the accuracy and precision in quantitative phosphoproteomics. We have developed a targeted phosphorylation site specific label-free quantitation approach to obtain phosphorylation stoichiometry and also monitor their dynamics of during toll-like receptor mediated signaling. Proteins were derived from human embryonic kidney 293T cells stably expressing toll-like receptor 4 that were transiently transfected with genes encoding the protein of interest. HA-Flag-tagged proteins were pulled-down via immunoprecipitation with anti-flag peptide epitope of the antibody The IP was separated via SDS-PAGE. The gel-bands specific for the protein(s) of interest were excised, trypsinized and subjected to nano-liquid chromatography/mass spectrometry on an Eksigent 2D/ETD enabled LTQ-Orbitrap XL and Velos instruments. The data was searched using Mascot, Sequest, and Zcore search engines, phosphorylation was confidently localized using Ascore, and targeted label-free quantitation was done using a Perl script that was developed in-house for calculating the peak areas of select-ion chromatograms.