General Approach To Determine Disulfide Connectivity in Cysteine-Rich Peptides by Sequential Alkylation on Solid Phase and Mass Spectrometry.
Within the field of bioprospecting, disulfide-rich peptides are a promising group of compounds that has the potential to produce important leads for new pharmaceuticals. The disulfide bridges stabilize the tertiary structure of the peptides and often make them superior drug candidates to linear peptides. However, determination of disulfide connectivity in peptides with many disulfide bridges has proven to be laborious and general methods are lacking. This study presents a general approach for structure elucidation of disulfide-rich peptides. The method features sequential reduction and alkylation of a peptide on solid phase combined with sequencing of the fully alkylated peptide by tandem mass spectrometry. Subsequently, the disulfide connectivity is assigned on the basis of the determined alkylation pattern. The presented method is especially suitable for peptides that are prone to disulfide scrambling or are unstable in solution with partly reduced bridges. Additionally, the use of small amounts of peptide in the lowest nmol range makes the method ideal for structure elucidation of unknown peptides from the bioprospecting process. This study successfully demonstrates the new method for seven different peptides with two to four disulfide bridges. Two peptides with previous contradicting publications, μ-conotoxin KIIA and hepcidin-25, are included, and their disulfide connectivity is confirmed in accordance with the latest published results.
Norut Northern Research Institute , 9294 Tromsø, Norway.
Anastasia
A
Albert
Norut Northern Research Institute , 9294 Tromsø, Norway.
J Johannes
JJ
Eksteen
Department of Chemistry, Faculty of Science and Technology, UiT The Arctic University of Norway , 9037 Tromsø, Norway.
Johan
J
Isaksson
Norut Northern Research Institute , 9294 Tromsø, Norway.
Myagmarsuren
M
Sengee
Department of Pharmacy, Faculty of Health Sciences, UiT The Arctic University of Norway , 9037 Tromsø, Norway.
Terkel
T
Hansen
Norut Northern Research Institute , 9294 Tromsø, Norway.
Terje
T
Vasskog
D000478:Alkylation; D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004220:Disulfides; D010455:Peptides