Nuclear Magnetic Resonance seq (NMRseq): A New Approach to Peptide Sequence Tags.

David Wilson,Norelle L Daly

doi:10.3390/toxins10110437

Abstract

Structural analysis of peptides with nuclear magnetic resonance (NMR) spectroscopy generally relies on knowledge of the primary sequence to enable assignment of the resonances prior to determination of the three-dimensional structure. Resonance assignment without knowledge of the sequence is complicated by redundancy in amino acid type, making complete de novo sequencing using NMR spectroscopy unlikely to be feasible. Despite this redundancy, we show here that NMR spectroscopy can be used to identify short sequence tags that can be used to elucidate full-length peptide sequences via database searching. In the current study, we have used this approach to identify conotoxins from the venom of the cone snail Conus geographus and determined the three-dimensional structure of a member of the I3 superfamily. This approach is most likely to be useful for the characterization of disulfide-rich peptides, such as those that were chosen for this study, as they generally have well-defined structures, which enhances the quality of the NMR spectra. In contrast to other sequencing methods, the lack of sample manipulation, such as protease digestion, allows for subsequent bioassays to be carried out using the native sample used for sequence identification.

Highlights

Peptides are highly prevalent in nature and they are of particular interest as therapeutic and bioinsecticide leads due to their typically improved stability, ease of production, and reduced antigenicity over proteins [1,2,3,4]
This approach involves the analysis of standard two-dimensional nuclear magnetic resonance (NMR) spectra, including COSY, TOCSY, HSQC, and NOESY, and it is highly suited to the analysis of small peptides with well-resolved cross-peaks in the NMR spectra, such as venom peptides
Defense-evoked venom was milked from C. geographus and fractionated using semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC) (Figure 1A)

Summary

Introduction

Peptides are highly prevalent in nature and they are of particular interest as therapeutic and bioinsecticide leads due to their typically improved stability, ease of production, and reduced antigenicity over proteins [1,2,3,4]. Advances in omics technologies, such as genomics, transcriptomics, and proteomics have allowed for relatively rapid elucidation of vast numbers of peptide sequences through an integrated bioinformatic approach [5] None of these technologies allow for non-destructive determination of the sequence of the peptide in its native form. We introduce NMRseq, a complementary proteomics workflow, which allows for the non-destructive determination of sequence tags of a peptide in the native form, while simultaneously providing data on the three-dimensional structure of the molecule This approach involves the analysis of standard two-dimensional nuclear magnetic resonance (NMR) spectra, including COSY, TOCSY, HSQC, and NOESY, and it is highly suited to the analysis of small peptides with well-resolved cross-peaks in the NMR spectra, such as venom peptides.

Methods

Results

Discussion

Conclusion