Coupling Formic Acid Assisted Solubilization and Online Immobilized Pepsin Digestion with Strong Cation Exchange and Microflow Reversed-Phase Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry for Integral Membrane Proteome Analysis

摘要

In this study, a facile system for membrane proteomenprofiling was established, in which membrane proteins werensolubilized by formic acid, online digested by a pepsin-basednimmobilized enzyme reactor (pepsin-IMER), and analyzednby strong cation exchange and microflow reversed-phasenliquid chromatography with electrospray ionization tandemnmass spectrometry (SCX-μRPLC-ESI-MS/MS). Under optimizednconditions, such a system showed excellent compatibilitynbetween all crucial steps and was successfully appliednfor analyzing integral membrane proteins extracted from ratnliver microsomes. Out of the 235 unique proteins positivelynidentified, 39% (91/235) were annotated as membranenproteins with one or more transmembrane domains (TMDs).nIt is anticipated that the efficient sample treatment and thenrelevant online analytical system might provide a promisingntool for automated and comprehensive profiling of membranenproteomes.