Mass Spectrometry Analysis of Intact Proteins from Crude Samples

Abstract

Analysis of intact proteins by native mass spectrometry has emerged as a powerful tool for obtaining insight into subunit diversity, post-translational modifications, stoichiometry, structural arrangement, stability, and overall architecture. Typically, such an analysis is performed following protein purification procedures, which are time consuming, costly, and labor intensive. As this technology continues to move forward, advances in sample handling and instrumentation have enabled the investigation of intact proteins in situ and in crude samples, offering rapid analysis and improved conservation of the biological context. This emerging field, which involves various ion source platforms such as matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) for both spatial imaging and solution-based analysis, is expected to impact many scientific fields, including biotechnology, pharmaceuticals, and clinical sciences. In this Perspective, we discuss the information that can be retrieved by such experiments as well as the current advantages and technical challenges associated with the different sampling strategies. Furthermore, we present future directions of these MS-based methods, including current limitations and efforts that should be made to make these approaches more accessible. Considering the vast progress we have witnessed in recent years, we anticipate that the advent of further innovations enabling minimal handling of MS samples will make this field more robust, user friendly, and widespread.

High-Resolution Native Mass Spectrometry

“…We have described here the laborious efforts often needed to purify protein complexes of interest, aiming to desalt these analytes as much as possible, for instance by several rounds of dialysis, while simultaneously avoiding the copurification of detrimental detergent molecules and polymers. However, with the increased sensitivity and capabilities to desolvate ions within the latest mass analyzers, ,, several pioneering attempts have been made to analyze proteins and protein complexes directly from cellular broths or cellular membranes, and reasonable success has already been achieved. Such attempts will certainly expand and make native MS hopefully even more feasible also for nonexperts.…”

Structure-based design of CDC42 effector interaction inhibitors for the treatment of cancer

“…The two samples were buffer exchanged in 20 mM Hepes pH 7.5, 40 mM NaCl, 5 mM MgCl 2 , 1 mM DTT and loaded on a RESOURCE Q (Cytiva) column for anion exchange chromatography. The efficiency of nucleotide loading was evaluated by native state mass spectrometry ( Vimer et al, 2020 ; Geoghegan et al, 1999 ). The purified proteins were buffer exchanged in 10 mM ammonium acetate pH 6.8 and diluted to 3 μM in 10 mM ammonium bicarbonate pH 6.5 added with final 3% acetonitrile.…”

Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology

“…A second approach is native MS (nMS, Figure 1, middle), an MS-based technique that directly works on intact proteins and protein complexes by circumventing the denaturing step, thereby providing a bridge between interactomics and structural biology (for review, see Bolla et al, 2019;Vimer et al, 2020;Tamara et al, 2021). In nMS, proteins or protein complexes are first buffer-exchanged into a MS-compatible buffer that minimally disturbs their native structural organization, including inter-and intra-molecular connections.…”