Reversed-phase separation parameters for intact proteins using liquid chromatography with triple quadrupole mass spectrometry.

The separation of intact proteins is inherently more complex than that of small molecules using reversed-phase liquid chromatography. The goal of this work was to determine a reasonable set of operational parameters (a recommended starting point for other analysts) for the separation of intact proteins and their detection by triple quadrupole mass spectrometry. Although protein separations have been studied for many years, the direct detection of intact proteins with mass spectrometry requires special considerations of mobile phase additives to achieve efficient separation and sensitive detection. Myoglobin, cytochrome c, lactalbumin, lysozyme, and ubiquitin were used as model analytes to investigate chromatographic method development using a triple quadrupole mass spectrometer and detection by multiple reaction monitoring. Chromatographic parameters including the concentration of trifluoroacetic acid, flow rate, gradient slope, temperature, mobile phase composition, and stationary phase chemistry were evaluated. Protein charge state profiles were also monitored for temperature and modifier effects. An optimized method using 0.2 mL/min flow rate, 15% gradient slope, and 75°C with a combined trifluoroacetic acid and formic acid modified mobile phase was developed.