Experimental Assignment of Disulfide‐Bonds in Purified Proteins

Abstract

The formation of disulfide bonds in proteins is an important post‐translational modification that is critical for stabilizing the native structures of proteins, particularly proteins exposed to oxidizing environments. For this reason, most cysteines in secreted proteins or protein domains on the surface of the cell are in disulfides, whereas most cysteines in the cytoplasm are in the unmodified ‐SH form. Disulfide linkages must be experimentally determined, as they cannot be predicted from amino acid sequence. These assignments provide insights into three‐dimensional structure and contribute to the understanding of structural‐functional relationships. This unit details a series of protocols that have been applied successfully to map disulfide bonds in proteins. The general strategy involves chemical or proteolytic cleavage of the protein followed by chromatographic separation of the resultant peptides. Mass spectrometry is used to identify disulfide‐containing peptides and determine sites of disulfide linkage. A partial reduction and alkylation strategy for mapping disulfide linkages in peptides with multiple disulfide bonds is also presented. © 2019 by John Wiley & Sons, Inc.

In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI–MS spectrum

“…Although Cys-capping modifications protect the molecule from aggregation and scrambling mediated by inter-and intra-molecular disulfide bonds, respectively, it needs to be addressed if the final outcome is to use the unpaired Cys for further modification, for example, in a drug conjugation process [41,42]. Another issue also to be addressed is the potential protein heterogeneity if the final intention is the use of the dimer molecule through disulfide bonds [35,36,43,44]. 538-538 ) in Fig.…”

Hard nut to crack: Solving the disulfide linkage pattern of the Neosartorya (Aspergillus) fischeri antifungal protein 2

“…Despite the introduction of more and more sophisticated methods, accurate prediction of disulfide bonding patterns in cysteine‐rich proteins is still challenging (Márquez‐Chamorro & Aguilar‐Ruiz, 2015). In most cases, an experimental determination is essential to confirm the in silico results and to understand the impact of the disulfide bonding pattern on the structural‐functional relationships, especially, when a protein is considered as a potential template for future drug design (Tang & Speicher, 2019), such as fungal AFPs (Galgóczy et al, 2019). Crystal diffraction data analyses, NMR assignments with MS‐coupled proteolytic digestion methods proved to be appropriate to determine and confirm the disulfide connectivity in different AFPs possessing six cysteine residues, such as abcabc in P. chrysogenum PAF (Batta et al, 2009), PAFB (Huber et al, 2018), and N .…”

In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum

“…Although Cys-capping modifications protect the molecule from aggregation and scrambling mediated by inter-and intra-molecular disulfide bonds, respectively, it need to be adressed if the final outcome is to use the unpaired Cys for further modification as for example drug conjugation process (44,45). Another issue also to be adressed is the potential protein heterogeneity if the final intention is the use of the dimer molecule through disulphide bonds (38,39,46,47).…”