Shotgun analysis of integral membrane proteins facilitated by elevated temperature

The beneficial effects on peak selectivity and resolution of conducting liquid chromatography (LC) at elevated temperature (e.g., 30-80 degrees C) are generally well-known; however, its importance for peptide recovery is not nearly as well recognized. This report demonstrates that microLC analysis of membrane proteomic samples significantly benefits from the application of heat. Enriched membrane and membrane-embedded peptides (the latter obtained by membrane shaving) were analyzed by microLC-tandem mass spectrometry (MS/MS) from 20 to 60 degrees C using a standard reversed-phase material. Maximal protein and hydrophobic peptide recovery was obtained at 60 degrees C. The membrane-shaving method employed, a recently optimized version of the high pH/proteinase K protocol, provided significant integral membrane protein enrichment: 98% of identified proteins were predicted to have at least one transmembrane domain (87% to have at least three), and 68% of peptides were predicted to contain transmembrane segments. Analysis of this highly enriched sample at elevated temperature increased protein identifications by 400%, and peptide identifications by 500%, as compared to room-temperature separation. Given that most microLC-MS/MS analyses are currently conducted at room temperature, the findings described herein should be of considerable value for improving the comprehensive study of integral membrane proteins.