Preanalytical Strategies for Native Mass Spectrometry Analysis of Protein Modifications, Complexes, and Higher-Order Structures<b></b>

Proteins are essential biological macromolecules that play key regulatory roles in all biological processes. Abnormalities in these processes are often reflected in proteins, manifesting as changes in their structure, sequence, folding state, stoichiometry, or spatial and temporal distribution. Proteins serve as biological targets for drugs and other therapeutics and can also function as therapeutic agents to restore normal biological functions by treating diseases. Hence, it is essential to study native protein species, their modifications, higher-order structures, and complexes, which can be extremely difficult due to the challenges in preserving their native conditions and the instrumental capability required for such analysis. High-resolution mass spectrometry (HRMS) instruments provide advanced technical capabilities to study intact protein species from their gas phase ions after the protein solution is sprayed into the mass spectrometers. However, there are debates about the gas-phase protein structures obtained through mass spectrometry and the resemblance to their biological native state. This review discusses various techniques for isolating, separating, and enriching intact protein species for their native mass spectrometry (nMS) analysis. Emerging technologies, such as automated sample preparation, ion mobility spectrometry, and ambient surface mass spectrometry, are briefly discussed. This review aims to serve as a general guideline for beginners, primarily focusing on the preanalytical strategies and critical instrument parameters for nMS analysis of intact proteins, proteoforms, protein complexes, and higher-order structures.