Mass Spectrometric Profiling of Protein Complexes

Eluted proteins were separated on 4–12% gradient SDS-PAGE gels (Invitrogen) and stained with Colloidal Coomassie Blue. Each gel lane was cut into 23 equal gel slices and proteins therein were in-gel digested with trypsin as described57 (link). Tryptic peptides from each gel slice were analysed as described58 (link) by nanoflow HPLC (Agilent 1100, Agilent Technologies) coupled to nanoelectrospray LTQ-Orbitrap XL mass spectrometer (Thermo Fischer Scientific). Raw MS data files were analysed by MaxQuant (version 1.3.0.5)22 (link) and Andromeda59 (link) using the UniProt rat protein database (version 05.13) and with selected “label-free protein quantification” (LFQ). Results from MaxQuant were further processed using Perseus (version 1.3.0.4, www.perseus-framework.org). Contaminant and reverse entries were filtered and all LFQ intensities were logarithmised. Missing values were imputed with random numbers using normal distribution (width = 0.3, down shift = 1.8) to simulate low abundance intensity values21 (link). A modified t test (250 permutations, FDR = 0.01, S₀ = 2)60 (link)61 (link) was applied to identify proteins significantly enriched within each IP experiment using four biological replicates and compared to identical control IP.