Proteomic Analysis of olfactory cilia membranes

Membranes of olfactory cilia were isolated from mouse olfactory epithelia. Sample fractionation was carried out in parallel by two methods: Proteins were fractionated by SDS-PAGE, the gel lanes were cut into bands and the proteins digested in-gel. Alternatively, proteins were digested in solution and peptides fractionated by strong cation exchange chromatography. Samples were analyzed using an LTQ-Orbitrap Velos. Mass spectrometric data of all replicates were searched together using the software MaxQuant (version 1.2.0.18) and its integrated search engine Andromeda against the UniProt reference proteome set for Mus musculus (http://www.uniprot.org/taxonomy/complete-proteomes; version of March 2, 2012, containing 54,231 entries) appended with a list of common contaminant proteins provided by MaxQuant. Database searches were performed with tryptic specificity allowing two missed cleavages and 7 ppm initial mass tolerance for precursor ions and 0.5 Da for fragment ions. Oxidation of methionine was considered as variable modification; fixed modifications were not included. Peptides and proteins were identified with a false discovery rate of 0.01.