Authors:

Olivia B Williams

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Despite its infrequent occurrence in the proteome, cysteine is the most nucleophilic amino acid. Its involvement in numerous biological processes makes it extremely prominent in covalent therapy research. Cysteine's pKa is known to shift dramatically with changes in its microenvironment, ranging between 3-10, which presents an opportunity to target specific cysteines in the proteome for covalent drug development. This can be achieved by identifying novel proteoforms; these are the unique proteins that can arise from the same gene through post-translational modifications (PTMs), alternative splicing events, and mutations. Currently, cysteine reactivity profiling is studied through bottom-up proteomics, but this approach suffers from a loss of structural information, causing the loss of the connection between reactivity and PTMs, and making it difficult to localize differentially reactive cysteines. In this study, we aim to better characterize proteoforms with differentially reactive cysteines using a combination of protein-level enrichment and top-down mass spectrometry. To test this, we will use activity-based protein profiling coupled with top-down liquid chromatography-mass spectrometry. These techniques allow us to isolate, label, identify, and characterize proteoforms containing reactive cysteines in complex samples. Preliminary experiments were analyzed via in-gel fluorescence and Coomassie stain and demonstrated comparable labeling efficiency between iodoacetamide, the standard for cysteine labeling, and a cell- permeable maleimide derivative. Future experiments will aim to evaluate the enrichment method for efficiency before moving into competitive experiments to determine the presence and placement of reactive cysteines within more complex samples. Keywords: Cysteine Reactivity; Proteomics; Activity-Based Protein Profiling (ABPP); Proteoform; Post-Translational Modifications (PTM)

Source:

Purdue University / 2025

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Co-authors:

Olivia B Williams