Strategies in mass spectrometry for the assignment of Cys-Cys disulfide connectivities in proteins

Abstract

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.

Keywords::

• cysteines
• disulfide
• mass spectrometry
• proteins
• proteomics
• venom

Acknowledgements

The authors thank S Schmid, head of the Institute of Life Technologies, HES-SO Valais and gratefully acknowledge the support of the HES-SO Valais.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Key issues

• • Disulfides are crucial to the structure and function of proteins.

• • Venom proteins are increasingly prominent in drug discovery. They are rich in disulfides and frequently possess a tightly disulfidebridged core with closely spaced or vicinal cysteines lacking enzymatic cleavage sites between them. Thus, they present a challenge for disulfide assignment.

• • Disulfide bridge scrambling occurs at alkaline pH, as is optimal for many proteolytic enzymes. Sample preparation should include blocking of free thiol groups and/or be performed at an acidic pH.

• • Disulfide bridges can be assigned using mass spectrometry (MS) by measuring the masses of unreduced proteolytic fragments bonded by a single disulfide.

• • Chemical cleavage and partial reduction of disulfides are strategies for sample preparation when enzymatic cleavage fails to produce peptides linked by a single disulfide.

• • Tandem MS can elucidate the connectivity of polypeptides linked with multiple disulfides, whose connectivity is ambiguous in MS1.

• • Tandem MS is used with fragmentation along the peptide bond and/or fragmentation of disulfides. This depends on instrumentation and experimental conditions such as collision energy.

• • Software development for data analysis has the potential to transform MS/MS-based disulfide connectivity determination in the future.

Notes