Isolation, Separation and Injection Strategies for Targeted Mass Spectrometry Analysis of Intact Protein under Native-Like Condition

Proteins are important biological macromolecules that have key regulatory roles in all biological processes and pathways. Hence, abnormalities in biological processes and pathways are often reflected in protein molecules in many ways including changes in their structure, sequence, folding state, stoichiometry, spatial and temporal distribution, among others. Proteins serve as biological targets of drugs and other therapeutic agents, and they can also function as therapeutic agents themselves to help restore normal biological functions and treat disease conditions. Hence, it is important to have the ability to study how they exist in native conditions, 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, isolating an intact protein from biological matrix at sufficient quantity and resolving its various proteoforms while preserving the native state until it reaches the mass spectrometer presents significant analytical challenges. This review article discusses various techniques for isolating, separating, and injecting intact proteins for targeted analysis by native mass spectrometry (nMS). Additionally, specialized mass spectrometry technologies such as ambient surface mass spectrometry, and matrix assisted laser desorption mass spectrometry as well as gas phase separation such as ion mobility spectrometry are briefly discussed. The goal of this review is to provide a comprehensive resource by systematically outlining and discussing sample preparation and injection techniques for nMS analysis of intact proteins via electrospray ionization, offering examples to guide the readers entering this field. Topics such as mass spectrometry hardware configuration, fragmentation techniques, and data analysis are not covered in this review, as they are extensively discussed elsewhere in the literature.