Successful research focusing on antibody and drug interactions requires preservation of non-covalent interactions, maintenance of high-degree folding, and increased mass separation and signal. To achieve this analysis, it is important that a protein stay in a native condition without the use of organic solvents. In the absence of organic solvents, electrospray produces larger droplets, making for less efficient desolvation. Additionally, scanning must take place around 8,000 m/z to observe results of native antibody samples. This type of analysis not only lets the user see antibody and drug interactions but also allows this to occur in the presence of other antibodies and drugs. Currently, this type of analysis is not available on commercial quadrupole-Orbitrap mass spectrometer instruments; however, Scheffer et al. show that with some modifications this analysis is possible.1
The group expanded the modes of sample workflow by adding a high mass range (HMR) mode to the existing options of standard mode and protein mode. The difference in modes begins in sample preparation, where standard mode requires a sample to undergo trypsin digest. It uses an Acclaim C18 column on a Vanquish ultra-high-performance liquid chromatography system (both Thermo Scientific). Protein mode digests sample using FabRICATOR digest, which allows for antibody subunit fragmentation, followed by the use of a MAbPac reversed phase column (Thermo Scientific) during liquid chromatography. HMR mode allows the sample to remain in a native or denatured state during analysis, where native samples use a MAbPac size exclusion column and denatured samples use a MAbPac reversed phase column for liquid chromatography separation. Mass spectrometry for these studies used a Q Exactive Plus mass spectrometer and a Q Exactive HF mass spectrometer with the BioPharma option (all Thermo Scientific).
Changes in high energy collision dissociation (HCD) mode include reduced C-trap and HCD cell gas pressures. Additionally, by trapping ions in the HCD cell before mass analysis, the protein ion can give increased signals in resolved samples. Other factors that the authors noted as being important to support desolvation and declustering include capillary and probe heater settings. To test the changes in protocol, the authors used intact trastuzumab (a monoclonal antibody used in the treatment of breast cancer) in both native and denatured conditions. In analyzing the results, they saw nearly identical spectra between the two preparations after deconvolution.
The HMR mode has several benefits. First, it offers users mass detection up to 8,000 m/z. Next, it expands the usability of the BioPharma platform to encompass three useful modes. Finally, in the HMR mode, the ion transfer and trapping have been improved to allow for sensitivity in resolution settings as high as 70,000. The HMR mode gives scientists an opportunity to study complex protein interactions in their native state—a useful tool in drug development and personalized medicine.
1. Scheffler, K., et al., (2016, June) “Enabling mass spectrometric analysis of intact proteins in native conditions on a hybrid quadrupole-orbitrap mass spectrometer,” poster presented at the 64th ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, June 5–9, 2016.