Protein kinases integrate cellular signals through complex phosphorylation cascades, yet resolving how chemical perturbations trigger and modulate these cascades in therapeutic targets remains a major...

This study advances top-down individual ion mass spectrometry (I2MS) to profile intact phospho-proteoforms of β- and α-catenins (85–110 kDa) within cadherin–catenin complexes. By modulating actomyosi....

Modalities and applications of targeted protein degradation (TPD) have rapidly expanded the therapeutic possibilities of proximity induced pharmacology. Here, Krone et al. spotlight three focal points...

Top-down proteomics is the study of intact proteins and their post-translational modifications with mass spectrometry. Historically, this field is more challenging than its bottom-up counterpart because the species are much bigger and have a larger number of possible combinations of sequences and modifications; thus, there is a great need for technological development. With improvements in instrumentation and a multiplicity of fragmentation modes available, top-down proteomics is quickly gaining in popularity with renewed attention on increasing confidence in identification and quantification. Here, we systematically evaluated the Sciex ZenoTOF 7600 system for top-down proteomics, applying standards in the field to validate the platform and further experimenting with its capabilities in electron-activated dissociation and post-translational modification site localization. The instrument demonstrated robustness in standard proteins for platform QC, as aided by zeno trapping. We were also able to apply this to histone post-translational modifications, achieving high sequence coverage that allowed PTM’s site localization across protein sequences with optimized EAD fragmentation. We demonstrated the ability to analyze proteins spanning the mass range and included analysis of glycosylated proteins. This is a reference point for future top-down proteomics experiments to be conducted on the ZenoTOF 7600 system.

Orbitrap-based individual ion mass spectrometry enables charge detection mass spectrometry application on a broadly accessible mass spectrometry platform, enabling the analysis of complex mixtures tha...

This paper presents detailed results of previously reported protein studies conducted using a prototype multi-reflecting time-of-flight mass spectrome…

Top-down proteomics, the characterization of intact proteoforms by tandem mass spectrometry, is the principal method for proteoform characterization in complex samples. Top-down proteomics relies on p...

The crucial roles of proteoforms in biological processes and disease mechanisms have been increasingly recognized. However, the rate at which new proteoforms are being discovered using top-down proteomics has far outpaced the rate at which the functional significance of different proteoforms can be determined. Because of the close connection between protein folding and protein function, protein folding stability measurements on proteoforms have the potential to identify functionally significant proteoforms of a given protein. While a number of mass spectrometry-based proteomics methods for making protein folding stability measurements on the proteomic scale have been reported over the past decade, none have been interfaced with top-down proteomics. Described here is a top-down (TD) stability of proteins from the rates of oxidation (SPROX) approach for making proteoform specific folding stability measurements. This approach is validated using a mixture of three model proteins with well-characterized protein folding behavior by conventional SPROX as well as other more conventional biophysical techniques. The method is also used to evaluate the relative folding stabilities of the <30 kDa protein fraction isolated from an MCF-7 cell lysate. The relative folding stabilities of 150 proteoforms from 83 proteins were successfully characterized in the cell lysate analysis using the TD-SPROX approach.