Elucidation of Search Parameters for Q-Exactive to Maximize Protein Identifications at 1 False Discovery Rate Using Wild-Type and FoxJ1 Knock Out Mouse Brain Tissues

摘要

All MS/MS spectra were acquired in the Orbitrap, allowing MS/MS data to be searched 0.02 Da rather than the 0.6 Da setting used for LTQ-Orbitraps. Data searched with greater than a 5 ppm mass tolerance for the precursor ion resulted in a greater amount of deamidated peptides than what was detected in the -5 to 5 ppm range. Deamidated peptides in the 5 to 20 ppm range were inaccurately identified. This was determined by manual inspection of both the MS and MS/MS data of a subset of the peptides. For global label-free quantification studies, searching without Deamidation as a variable modification resulted in no statistical differences then with search with Deamidation. Final parameters include ± 5 ppm for precursor ions, ± 0.02 Da for MS/MS spectra, and Deamidation in no longer a search criteria. A total of 5832 proteins were detected between WT and KO mouse brain lysates. Candidate proteins are further being investigated for their role in the FoxJ1 Pathway. The FoxP1 is belongs to the family of Forkhead Box proteins especially.