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

Abstract

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.

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

“…Recently, Wang et al. has shown the use of LC with triple quadrupole (QqQ) MS for the top‐down analysis of intact proteins using multiple reaction monitoring (MRM) mode [13–15]. Källsten et al.…”

“…Reversed‐phase LC (RP‐LC) is one of the commonly used methods for efficient separation of intact proteins [13,17,18]. When combined with MS systems, volatile mobile phase additives (e.g.…”

Supercharging and multiple reaction monitoring of high‐molecular‐weight intact proteins using triple quadrupole mass spectrometry

“…Further work on the analysis of intact proteins by Wang et al investigated intact protein ion transmission effects in the triple quadrupole instrument, as well as generic conditions for intact protein separations by reversed‐phase liquid chromatography/mass spectrometry (LC/MS) . In the former, ion scattering, charge transfer, mass resolution, and MRM summation effects were evaluated using a variety of different collision gases (Ar, He, and N 2 ).…”

“…Significant differences in behavior for intact protein ions, relative to small‐molecule ions, in the gas phase were readily apparent, suggesting that further efforts should be given to optimization of triple quadrupole mass spectrometry instrumentation for MRM‐based intact protein analysis. The LC/MS‐based chromatographic methodology developed used both formic acid and a small amount of trifluoroacetic acid in the mobile phase to yield high‐efficiency separations. As with the previous study, all of these studies focused on the analysis of relatively low‐molecular‐weight proteins.…”

“…These days, it has become highly desirable to analyze antibody proteins, antibody–drug conjugates, and other higher molecular weight proteins, which can sometimes have a molecular weight exceeding 150 kDa . One way to potentially improve the ESI‐MS detection of high‐molecular‐weight proteins is to use supercharging agents .…”