Gel-free mass spectrometry analysis of Drosophila melanogaster heads

Here we describe a bottom-up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem-mass spectrometry (MS/MS) that relies on a complete solubilisation and digestion of proteins. An enriched membrane fraction was prepared using equilibrium density centrifugation on a discontinual sucrose gradient, followed by solubilisation using FASP method, tryptic and consequential chymotryptic digestion of proteins. Peptides were separated by reversed-phase (RP) LC at high pH in the first dimension and acidic RP-LC coupled directly to Orbitrap Velos Pro mass spectrometer.