Top-Down and Bottom-Up Proteomics Methods to Study RNA Virus Biology

Abstract

RNA viruses cause a wide range of human diseases that are associated with high mortality and morbidity. In the past decades, the rise of genetic-based screening methods and high-throughput sequencing approaches allowed the uncovering of unique and elusive aspects of RNA virus replication and pathogenesis at an unprecedented scale. However, viruses often hijack critical host functions or trigger pathological dysfunctions, perturbing cellular proteostasis, macromolecular complex organization or stoichiometry, and post-translational modifications. Such effects require the monitoring of proteins and proteoforms both on a global scale and at the structural level. Mass spectrometry (MS) has recently emerged as an important component of the RNA virus biology toolbox, with its potential to shed light on critical aspects of virus–host perturbations and streamline the identification of antiviral targets. Moreover, multiple novel MS tools are available to study the structure of large protein complexes, providing detailed information on the exact stoichiometry of cellular and viral protein complexes and critical mechanistic insights into their functions. Here, we review top-down and bottom-up mass spectrometry-based approaches in RNA virus biology with a special focus on the most recent developments in characterizing host responses, and their translational implications to identify novel tractable antiviral targets.

Proteomic Approaches to Unravel Mechanisms of Antibiotic Resistance and Immune Evasion of Bacterial Pathogens

“…However, this approach typically requires some form of charge state stabilization or extremely high m/z resolution from the instrument ( 44 ). Often, top-down approaches are combined with tandem MS methods to distinguish between protein species and various modifications can be both located and identified ( 41 ).…”

“…Regarding the topic of this review, in vivo XL-MS is a promising tool to discover virus–host associations and pathways ( 50 ), as well as potential interfaces for drug design or to derive a drug’s mode of action. With regards to drug discovery, nuclear magnetic resonance (NMR) and X-ray crystallography are still the structural methods of choice in a hit-to-lead or lead optimization strategy, however, native MS and hydrogen–deuterium exchange have been used as alternative methods and are regularly employed by biopharma companies ( 41 ).…”

“…Conversely, in top-down methods intact proteins are analyzed, making these workflows particularly suited for the analysis of distinct and co-existing post-translational modifications (PTMs) of a protein or protein complex ( 41 , 42 ). Some of the major advantages to top-down approaches include largely eliminating the problem of protein inference and preserving higher-level organizational information ( 43 ).…”

The kinetics of SARS-CoV-2 nsp7-11 polyprotein processing and impact on complexation with nsp16

“…Here, we aim to investigate the dynamics of in vitro processing of SARS-CoV-2 nsp7-11 with native mass spectrometry (MS). Native MS is a highly sensitive method allowing simultaneous monitoring of cleavage products making it very versatile to study dynamic and heterogenous processes 14 . Since in native MS, the natural folding and protein-protein interactions are preserved 15 , the protein complexes formed by the processing products can be detected.…”

Special Issue “Next-Generation Technologies to Understand Mechanisms of Virus Infections”

“…This group observed that the BRD4 inhibitors disrupted binding to acetylated transcriptional regulators. This study is complemented by a review by Simanjuntak et al [ 7 ] that describes novel MS tools to study the structure and composition of protein complexes, providing critical mechanistic insights into their functions. New approaches in top-down and bottom-up mass spectrometry-based are presented to identify novel- and tractable antiviral targets.…”