Fourier‐transform ion cyclotron resonance mass spectrometry for characterizing proteoforms

Abstract

Proteoforms contribute functional diversity to the proteome and aberrant proteoforms levels have been implicated in biological dysfunction and disease. Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS), with its ultrahigh mass-resolving power, mass accuracy, and versatile tandem MS capabilities, has empowered top-down, middle-down, and native MS-based approaches for characterizing proteoforms and their complexes in biological systems. Herein, we review the features which make FT-ICR MS uniquely suited for measuring proteoform mass with ultrahigh resolution and mass accuracy; obtaining in-depth proteoform sequence coverage with expansive tandem MS capabilities; and unambiguously identifying and localizing post-translational and noncovalent modifications. We highlight examples from our body of work in which we have quantified and comprehensively characterized proteoforms from cardiac and skeletal muscle to better understand conditions such as chronic heart failure, acute myocardial infarction, and sarcopenia. Structural characterization of monoclonal antibodies and their proteoforms by FT-ICR MS and emerging applications, such as native top-down FT-ICR MS and high-throughput top-down FT-ICR MS-based proteomics at 21 T, are also covered. Historically, the information gleaned from FT-ICR MS analyses have helped provide biological insights. We predict FT-ICR MS will continue to enable the study of proteoforms of increasing size from increasingly complex endogenous mixtures and facilitate the benchmarking of sensitive and specific assays for clinical diagnostics.

Novel Strategies to Address the Challenges in Top-Down Proteomics

“…Top-down MS was first coined by McLafferty and co-workers in 1999, and the early top-down MS studies generally took a targeted approach focusing on offline purified single proteins or simple protein mixtures due to the inherent limitations of intact protein analysis. − Top-down proteomics, pioneered by Kelleher and co-workers, includes the front-end fractionation of intact proteins, high-resolution MS, and back-end informatics, making it possible to identify proteins from complex mixtures. ,, In the past decade, top-down proteomics has experienced rapid growth due to many technological advances, − thereby enabling the applications of top-down proteomics to complex protein mixtures and for understanding basic biological functions, unraveling disease mechanisms, and discovering new biomarkers. − However, many challenges still exist for top-down MS-based proteomics, especially protein solubility, dynamic range, proteome complexity, data analysis, proteoform–function relationships, and analytical throughput of top-down proteomics for precision medicine (Figure ). Previous reviews on top-down proteomics have already described various aspects of top-down proteomics such as technological advancements and biomedical applications, among others. − ,,,− In this Account & Perspective, we will focus on the most pressing challenges currently facing the top-down proteomics field. We illustrate how recent and future technological innovation could help address these challenges to advance top-down proteomics toward the mainstream.…”

1DE-MS Profiling for Proteoform-Correlated Proteomic Analysis, by Combining SDS-PAGE, Whole-Gel Slicing, Quantitative LC–MS/MS, and Reconstruction of Gel Distributions of Several Thousands of Proteins

“…Protein solubility represents a major challenge in top-down MS-based proteomics because efficient extraction and solubilization of membrane proteins need strong detergents that are generally incompatible with MS and the removal often causes protein loss and hampers the analysis reproducibility, while MS-compatible detergents are often relatively mild with limited solubilization ability; (iii) 1DE-MS profiling can achieve high proteome coverage with the detection of several thousand proteins, while the numbers of proteins (not proteoforms) detected in top-down proteomics were mostly around several hundred (or less) with eukaryotic samples, still a fraction of the total proteins in complete proteomes. Top-down MS-based technology has proven to be a premier technology in proteoform analysis (progress and challenges have recently been well reviewed), − but considering the vast number of proteoforms that exist in biological systems and the complexity of most biological samples, it is important to integrate information obtained from different methods. , (3) Western blotting is a standard method that is widely used to detect proteins or proteoforms of interest or to validate detection results obtained with other methods (e.g., proteomics). As both methods use SDS-PAGE for protein separation, comparison between Western blots and 1DE-MS profiles can be done in a straightforward manner and provides validation or complementary information.…”

“…By measuring accurate masses of intact proteoforms with high- or ultrahigh-resolution MS, top-down proteomics could characterize proteoforms and identify PTMs and amino acid sequence variations. However, there are still many challenges to be addressed in this field, especially on protein size, protein solubility, proteome complexity, dynamic range, data analysis, and analytical throughput. − …”

Fourier transform ion cyclotron resonance mass spectrometry at the true cyclotron frequency

“…Comprehensive protein structural analysis is one of the most important FT-ICR MS applications nowadays (Tucholski & Ge, 2021). To demonstrate the ability of performing protein analysis at the true cyclotron frequency, Figure 9 demonstrates the broadband frequency spectrum of an 8.6-kDa protein, ubiquitin, obtained with a 2xNADEL ICR cell on a 9.4 T FT-ICR MS.…”