Comparison of Proteome Discoverer and PEAKS Studio for Phosphoproteome Analysis

摘要

Phosphorylation of proteins is a reversible post-translational modification that is fundamental in the regulation of cellular processes, such as protein function, cellular signaling and protein-complex formation. The ability to perform a global analysis of phosphoproteome will give us insight as to how mechanisms of proteins and pathways might become altered due to changing experimental conditions. Because only a fraction of proteins or any given protein is phosphorylated, enrichment of sample for phosphoproteins or phosphopeptides is essential to the success of identification. Briefly, 100 ug of mouse whole cell lysate was digested in-solution with trypsin overnight. Samples were cleaned-up using PepClean columns, and then processed using TiO2 Phosphopeptide Enrichment and Clean-up Kit (Pierce, Inc.). The enriched samples were injected onto nano-LC-LTQ Orbitrap with ETD in configuration of HCD and ETD as two microscans complementing information from MS/MS spectra. The acquisition method was created in data-dependent mode with one precursor scan in the Orbitrap, followed by fragmentation of the 4 most abundant peaks in both ETD detected in the LTQ, and in HCD detected in the Orbitrap. Tolerances were set to 10 ppm for the Orbitrap precursors and fragments while 0.8 Da for the ETD fragments. The following dynamic modifications were applied: Phospho/+79.966 Da (S, T, Y), Oxidation/+15.995 Da (M), Carboxymethyl / +58.005 Da (C). A Decoy database was also searched and a target FDR set for 0.01. For protein identification and localization of phosphorylated sites we used two software packages, Proteome Discoverer and PEAKS Studio. We present here a comparison of the search output obtained from these two software packages.