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Elucidating disulfide linkage patterns is a crucial part of protein characterization, for which mass spectrometry (MS) is now an indispensable analytical tool. In many cases, MS-based disulfide connectivity assignment is straightforwardly achieved using one-step protein fragmentation in the unreduced form followed by mass measurement of bridged fragments. By contrast, venom proteins, which are receiving increasing interest as potential therapeutics, are a challenge for MS-based disulfide assignment due to their numerous closely spaced cysteines and knotted disulfide structure, requiring creative strategies to determine their connectivity. Today, these include the use of an array of reagents for enzymatic and/or chemical cleavage, partial reduction, differential cysteine labeling and tandem MS. This review aims to describe the toolkit of techniques available to MS users approaching both straightforward and complex disulfide bridge assignments, with a particular focus on strategies utilizing standard instrumentation found in a well-equipped analytical or proteomics laboratory.

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