JOURNAL ARTICLE

Facilitating protein disulfide mapping by a combination of pepsin digestion, electron transfer higher energy dissociation (EThcD), and a dedicated search algorithm SlinkS

Fan Liu, Bas van Breukelen, Albert J R Heck
Molecular & Cellular Proteomics: MCP 2014, 13 (10): 2776-86
24980484
Disulfide bond identification is important for a detailed understanding of protein structures, which directly affect their biological functions. Here we describe an integrated workflow for the fast and accurate identification of authentic protein disulfide bridges. This novel workflow incorporates acidic proteolytic digestion using pepsin to eliminate undesirable disulfide reshuffling during sample preparation and a novel search engine, SlinkS, to directly identify disulfide-bridged peptides isolated via electron transfer higher energy dissociation (EThcD). In EThcD fragmentation of disulfide-bridged peptides, electron transfer dissociation preferentially leads to the cleavage of the S-S bonds, generating two intense disulfide-cleaved peptides as primary fragment ions. Subsequently, higher energy collision dissociation primarily targets unreacted and charge-reduced precursor ions, inducing peptide backbone fragmentation. SlinkS is able to provide the accurate monoisotopic precursor masses of the two disulfide-cleaved peptides and the sequence of each linked peptide by matching the remaining EThcD product ions against a linear peptide database. The workflow was validated using a protein mixture containing six proteins rich in natural disulfide bridges. Using this pepsin-based workflow, we were able to efficiently and confidently identify a total of 31 unique Cys-Cys bonds (out of 43 disulfide bridges present), with no disulfide reshuffling products detected. Pepsin digestion not only outperformed trypsin digestion in terms of the number of detected authentic Cys-Cys bonds, but, more important, prevented the formation of artificially reshuffled disulfide bridges due to protein digestion under neutral pH. Our new workflow therefore provides a precise and generic approach for disulfide bridge mapping, which can be used to study protein folding, structure, and stability.

Full Text Links

Find Full Text Links for this Article

Discussion

You are not logged in. Sign Up or Log In to join the discussion.

Related Papers

Remove bar
Read by QxMD icon Read
24980484
×

Save your favorite articles in one place with a free QxMD account.

×

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"