Elucidating the Structure of Cyclotides by Partial Acid Hydrolysis and LC−MS/MS Analysis
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- 作者：Siu Kwan Sze ; Wei Wang ; Wei Meng ; Randong Yuan ; Tiannan Guo ; Yi Zhu ; James P. Tam
- 刊名：Analytical Chemistry
- 出版年：2009
- 出版时间：February 1, 2009
- 年：2009
- 卷：81
- 期：3
- 页码：1079-1088
- 全文大小：295K
- 年卷期：v.81,no.3(February 1, 2009)
- ISSN：1520-6882
文摘
We describe here a rapid method to determine the cyclic structure and disulfide linkages of highly stable cyclotides via a combination of flash partial acid hydrolysis, LC−MS/MS, and computational tools. Briefly, a mixture of closely related cyclotides, kalata B1 and varv A purified from Viola yedoensis was partially hydrolyzed in 2 M HCl for 5 min by microwave-assisted hydrolysis or for 30 min in an autoclave oven (121 °C and 15 psi). The partially hydrolyzed peptide mixture was then subjected to LC−MS/MS analysis, with the disulfide linked-peptides fragmented by collision activated dissociation (CAD). A computer program written in-house (available for download at http://proteomics.sbs.ntu.edu.sg/cyclotide_SS) was used for interpreting LC−MS/MS spectra and assigning the disulfide bonds. Time-point analysis of single-disulfide fragments revealed that nonrandom acid catalyzed fragmentation mostly occurred at the turns which are solvent-exposed and often contain side chain functionalized amino acids such as Asx/Glx and Ser/Thr. In particular, the most susceptible bond for acid hydrolysis in kalata B1 and varv A was found to be the highly conserved N25−G26 which is also the head-to-tail ligation site of the linear precursor proteins, indicating that formation of the three disulfide bonds might precede cyclic structure closure by N25−G26 ligation. This observation is consistent with the recent report that the N25−G26 bond formation is the last step in the cyclotide biosynthetic pathway. The process demonstrated here can potentially be a high throughput method that is generally applicable to determine disulfide bonds of other relatively low-abundance cyclotides.