High-Resolution Native Mass Spectrometry

Abstract

Native mass spectrometry (MS) involves the analysis and characterization of macromolecules, predominantly intact proteins and protein complexes, whereby as much as possible the native structural features of the analytes are retained. As such, native MS enables the study of secondary, tertiary, and even quaternary structure of proteins and other biomolecules. Native MS represents a relatively recent addition to the analytical toolbox of mass spectrometry and has over the past decade experienced immense growth, especially in enhancing sensitivity and resolving power but also in ease of use. With the advent of dedicated mass analyzers, sample preparation and separation approaches, targeted fragmentation techniques, and software solutions, the number of practitioners and novel applications has risen in both academia and industry. This review focuses on recent developments, particularly in high-resolution native MS, describing applications in the structural analysis of protein assemblies, proteoform profiling ofamong othersbiopharmaceuticals and plasma proteins, and quantitative and qualitative analysis of protein–ligand interactions, with the latter covering lipid, drug, and carbohydrate molecules, to name a few.

Probing Affinity, Avidity, Anticooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses

“…In contrast to the solution-based measurements of MP, CD-MS measurements are performed on particles following their ionization and introduction into the gas phase. Over the years, a body of evidence has been accumulated demonstrating that protein complexes can be mass analyzed in the gas phase while generally retaining their native stoichiometry and other aspects of higher-order structure . The similar results here obtained by MP and native CD-MS support the validity of the presented findings and the absence of measurement biases.…”

A perspective toward mass spectrometry-based de novo sequencing of endogenous antibodies

“…Furthermore, in MS analysis, mass accuracy and resolution typically diminish with increasing molecular weight, even when using the latest high-resolution mass spectrometers. 30 , 71 , 72 In addition, full sequence coverage is generally unattainable for intact proteins with masses above 20 kDa. These factors have hindered implementation of protein-centric MS for de novo sequencing of antibodies.…”

Micro-flow Size-Exclusion Chromatography for enhanced native Mass Spectrometry of proteins and protein complexes

“…Recent applications of nMS have highlighted that additional information can be extracted on conformation or folding, topology, and dynamics of various protein complexes, ranging from smaller oligomeric complexes up to intact virus capsids and chaperone complexes 7 . The most-pivotal challenge of nMS is to transfer the higher-order structures and non-covalent complexes to the gas phase without loss of their native quaternary structure 8,9 . There are critical aspects associated with the sample preparation, ionization conditions, and instrumentation to ensure preservation of the structure during ionization 8,9 .…”

“…The most-pivotal challenge of nMS is to transfer the higher-order structures and non-covalent complexes to the gas phase without loss of their native quaternary structure 8,9 . There are critical aspects associated with the sample preparation, ionization conditions, and instrumentation to ensure preservation of the structure during ionization 8,9 . However, analysis of proteins with MS requires highly purified samples.…”

“…A lower flow rate is known to produce smaller charged droplets during the spraying process 15,16 . This may significantly improve the efficiency of the ionization, especially of that of larger biomolecules and protein complexes 7,9,14 . It may also offer a higher tolerance for salt contaminations and it may lead to reduced signal-suppression effects 7 .…”