Abstract
Due to their low abundance and limited number of trypsin cleavage sites, integral membrane proteins are notoriously difficult to identify and to analyze by mass spectrometry. Our approach to this problem is based on a novel technology for peptide sample preparation that uses plates with pre-deposited matrix spots of sub-micron size CHCA crystals prepared by vacuum sublimation onto an ultraphobic surface (Mass·Spec·Turbo Chips, QIAGEN). Such a design enables highest sensitivity analysis and reproducible and automatable sample preparation, suitable for both gel- and LC-based MALDI-MS approaches. In order to compare the chips’ performance with that of standard stainless steel target plates, different sample amounts of two model membrane proteins—bacteriorhodopsin and subunit A of ATP synthase—were separated by SDS-PAGE and in-gel digested with trypsin. After digestion, aliquots were spotted either as classical dried droplet preparations on stainless steel plates using CHCA as matrix, or applied directly onto the matrix spots pre-deposited on the chip surfaces. Spectra were acquired on Voyager DE-Pro or 4800 MALDI-TOF instruments (Applied Biosystems) and protein identification was performed by peptide mass fingerprint or MS/MS ion search using the Mascot algorithm (Matrix Science). The results were evaluated by comparing S/N ratios, sequence coverage, Mascot scores and the total ratio of protein identifications vs. unsuccessful attempts. Analyses of the spectra obtained revealed a significantly larger number of peptide signals using the chip approach, which resulted in increased sequence coverage and more reliable protein identifications.