Chapter title |
Peptidomic Identification of Cysteine-Rich Peptides from Plants
|
---|---|
Chapter number | 26 |
Book title |
Peptidomics
|
Published by |
Humana Press, New York, NY, February 2018
|
DOI | 10.1007/978-1-4939-7537-2_26 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7536-5, 978-1-4939-7537-2
|
Authors |
Xinya Hemu, Aida Serra, Dina A. Darwis, Tobias Cornvik, Siu Kwan Sze, James P. Tam |
Abstract |
Plant cysteine-rich peptides (CRPs) constitute a majority of plant-derived peptides with high molecular diversity. This protocol describes a rapid and efficient peptidomic approach to identify a whole spectrum of CRPs in a plant extract and decipher their molecular diversity and bioprocessing mechanism. Cyclotides from C. ternatea are used as the model CRPs to demonstrate our methodology. Cyclotides exist naturally in both cyclic and linear forms, although the linear forms (acyclotide) are generally present at much lower concentrations. Both cyclotides and acyclotides require linearization of their backbone prior to fragmentation and sequencing. A novel and practical three-step chemoenzymatic treatment was developed to linearize and distinguish both forms: (1) N-terminal acetylation that pre-labels the acyclotides; (2) conversion of Cys into pseudo-Lys through aziridine-mediated S-alkylation to reduce disulfide bonds and to increase the net charge of peptides; and (3) opening of cyclic backbones by the novel asparaginyl endopeptidase butelase 2 that cleaves at the native bioprocessing site. The treated peptides are subsequently analyzed by liquid chromatography coupled to mass spectrometry using electron transfer dissociation fragmentation and sequences are identified by matching the MS/MS spectra directly with the transcriptomic database. |
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Unknown | 6 | 40% |