Mouse microglia using High-pH fractionation and LC-MS/MS

ABSTRACT: To build a reference proteome, we established a BV-2 microglial proteome to a depth of 5494 unique protein groups using a novel strategy that combined FASP, StageTip-based high pH fractionation, and high-resolution mass spectrometry quickly and cost-efficiently. By bioinformatics analysis, the BV-2 proteome is a valuable resource for studies of microglial function, such as in the immune response, inflammatory response, and phagocytosis. Raw files were processed in MaxQuant version 1.2.2.5 and the Andromeda search engine against the IPI mouse database (version 3.87, 59,534 entries) containing both forward and reverse proteins sequences. Carbamidomethylation of cysteines was set as the fixed modification. Oxidation of methionine and acetylation of protein N-term was employed as a variable modification. The first search tolerance was set to 20 ppm, followed by a main search tolerance of 6 ppm. HCD fragment ion mass tolerance was set to 20 ppm. Peptides with a minimum of 6 amino acids were considered for identification. The false discovery rate (FDR) for all peptides, modification sites, and protein identifications were set to 0.01. Gene ontology of identified proteins at FDR < 1% was annotated using the DAVID bioinformatics resource tool and UniprotKB database. Pathway analysis was performed using the KEGG pathway database and Panther pathway database.

REANALYSED by: PAe005146

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Dohyun Han  

LAB HEAD: Dohyun Han

PROVIDER: PXD000168 | Pride | 2013-09-11

REPOSITORIES: Pride