Computational protocol: Differential Phagocytic Properties of CD45low Microglia and CD45high Brain Mononuclear Phagocytes—Activation and Age-Related Effects

Similar protocols

Protocol publication

[…] Raw data files were analyzed using MaxQuant v1.5.2.8 with Thermo Foundation 2.0 for RAW file reading capability, as previously published (, ). The search engine Andromeda was used to build and search a concatenated target-decoy IPI/Uniprot mouse reference (downloaded August 14, 2015). Protein Methionine oxidation (+15.9949 Da), and protein N-terminal acetylation (+42.0106 Da) were variable modifications (up to five allowed per peptide); cysteine was assigned a fixed carbamidomethyl modification (+57.0215 Da). Only fully tryptic peptides were considered with up to two mis-cleavages in the database search. A precursor mass tolerance of ±10 ppm was applied prior to mass accuracy calibration and ±4.5 ppm after internal MaxQuant calibration. Other search settings included a maximum peptide mass of 6,000 Da, a minimum peptide length of six residues, and 0.6 Da Tolerance for ion-trap HCD MS/MS scans. Co-fragmented peptide search was enabled to deconvolute multiplex spectra. The false discovery rate for peptide spectral matches, proteins, and site decoy fraction were all set to 1%. Quantification settings were as follows: re-quantify with a second peak finding attempt after protein identification has completed; match full MS1 peaks between runs; a 1.5-min retention time match window was used after an alignment function was found with a 20-min RT search space. The label-free quantification (LFQ) algorithm in MaxQuant (, ) was used for protein quantitation. Proteins with more than 25 percent overall missing data or more than 1 missing data point per treatment group were excluded from analysis. […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Application MS-based untargeted proteomics
Organisms Mus musculus