Nuno Bandeira, Department of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA
Julio Ng, Department of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA
Wei-Ting Liu, Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, USA
Majid Ghassemian, Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, USA
Thomas L Simmons, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California, USA.
William H Gerwick, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA
Roger Linington, Department of Chemistry, University of California Santa Cruz, Santa Cruz, California, USA.
Pieter C Dorrestein, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA
Pavel A Pevzner, Department of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA |
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The classical ribosomal protein synthesis pathway is not the only mechanism for cells to assemble amino acids into peptides – the alternative Non Ribosomal Peptide Synthesis is performed by large multi-enzyme complexes that represent both the biosynthetic machinery and the mRNA-free template for the biosynthesis of secondary metabolites. Non-Ribosomal Peptides (NRPs) are not directly inscribed in the genomic DNA and thus cannot be inferred with traditional DNA-based sequencing techniques. NRPs are of tremendous pharmacological importance and include antibiotics (penicillin, cephalosporine, vancomicine, etc.), immunosuppressors (cyclosporin), antiviral agents (luzopeptin A), antitumor agents (bleomycin), toxins (thaxtomin), and many peptides with yet unknown functions. Nevertheless, there is currently no technology capable of high-throughput sequencing of NRPs.
A fundamental difference between NRPs and common proteomics peptides is that NRPs are often cyclic peptides, single-handedly rendering impotent the battery of computational approaches designed for identification of linear peptides. Making the problem even more challenging, NRPs often incorporate non-standard and sometimes novel unknown amino acids. Thus, the standard set of 20 amino acid masses implicitly assumed by proteomics tools cannot be used for sequencing NRPs. We show how multi-stage mass spectrometry (MSn) can be combined with alignment-based algorithms for dereplication and de novo sequencing of cyclic NRPs. While our dereplication approach capitalizes on databases of known compounds to identify identical or related NRPs, the de novo sequencing approach allows for the characterization of previously unknown NRPs directly from MSn data.
We demonstrate our approach on a previously unknown cyclic NRP and on several known NRPs including Seglitide (a somatostatin receptor) and a mixture of Bacillus brevis Tyrocidine NRPs, the first antibiotic to be commercially available. Since mass spectrometry based analysis of NRPs is fast, inexpensive and uses minute amounts of sample, this automated approach opens the possibility of high-throughput dereplication and sequencing of many uncharacterized NRPs encountered in drug discovery efforts. |
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