Isolation, Separation and Injection Strategies for Native Mass Spectrometry Analysis of Intact Proteins

Proteins are important biological macromolecules that have key regulatory roles in all biological processes and pathways. Hence, abnormalities in biological processes and pathways are reflected on protein molecules in many ways including changes in their structure, sequence, folding, stoichiometry, spatial and temporal distribution, among others. Proteins are also the biological targets of drugs and other therapeutic agents and can also themselves be therapeutic agents to restore normal biological functions i.e. treat a disease conditions. Hence, it is important to have the ability to study their native structure, which can be onerous due to the challenges in preserving their native conditions as well as the instrumental capability required for such analysis. High resolution mass spectrometry instruments provide advanced technical capabilities to study intact protein molecules. However, extracting and isolating intact proteins from biological matrices at sufficient quantity while preserving their native state until it reaches the mass spectrometer presents significant analytical challenges. In this review article, various techniques that are available to isolate, separate and inject intact proteins for native mass spectrometry (nMS) analysis are discussed. Additionally, specialized NMS technologies such as hydrogen deuterium mass spectrometry, crosslinking mass spectrometry, and ambient surface mass spectrometry as well as gas phase separation such as ion mobility spectrometry are briefly discussed. The goal of this review is to create a resource by systematically enlisting and discussing various sample preparation and injection technologies for nMS analysis of intact proteins via electrospray ionization with examples which the readers can utilize as a guide before delving into this area. Topics such as mass spectrometry hardware configuration, fragmentation techniques, data analytics etc. are not discussed which are available in the literature.