Separation and identification of mouse liver membrane proteins using a gel-based approach in combination with 2DnanoLC-Q-TOF-MS/MS

The Thanh Tran and Van Chi Phan

Abstract

In this work, we present results of membrane proteome profiling from mouse liver tissues using a gel-based approach in combination with 2DnanoLC-Q-TOF-MS/MS. Following purification of the membrane fraction, SDS-PAGE was carried out as a useful separation step. After staining, gels with protein bands were cut, reduced, alkylated and trypsin-digested. The peptide mixtures extracted from each gel slice were fractionated by two-dimensional nano liquid chromatography (2DnanoLC) coupled online with tandem mass spectrometry analysis (NanoESI-Q-TOF-MS/MS). The proteins were identified by MASCOT search against a mouse protein database using a peptide and fragment mass tolerance of ±0.5 Da. Protein identification was carried out using a Mowse scoring algorithm with a confidence level of 95% and processed by MSQuant v1.5 software for further validation. In total, 318 verified membrane proteins from mouse liver tissues were identified; 66.67% of them (212 proteins) contained at least one or more transmembrane domains predicted by the SOSUI program and 43 were found to be unique microsome membranes. Furthermore, GRAVY values of membrane proteins varied in the range -1.1276 to 0.9016 and only 31 (9.76%) membrane proteins had positive values. The functions and subcellular locations of the identified proteins were categorized as well, according to universal GO annotations