File S1 - Sample Limited Characterization of a Novel Disulfide-Rich Venom Peptide Toxin from Terebrid Marine Snail Terebra variegata

This file contains Figure S1–Figure S9 and Table S1–Table S4. Figure S1, CAD of native Tv1. MS/MS spectrum recorded on a (M +2H)+2 ion after reduction of cysteine residues. The sequence is given above the spectrum and observed b, a and y-type fragment ions are labeled in the spectrum. Observed peptide backbone cleavage is indicated in the sequence above with and for N- and C-terminal fragment ions, respectively. Doubly charged fragment ions are labeled with +2. The neutral loss of water from the precursor ion is shown as [M+2H]+2–H20, but neutral losses of fragment ions are not labeled. The spectrum was recorded at a resolution of 7500 at m/z 400 and all fragment ions have a mass accuracy of better than 5 ppm. Figure S2, ETD of native (black) and synthetic Tv1 (blue). MS/MS spectrum recorded on a (M +6H)+6 ion after conversion of cysteine residues to dimethyl lysine analogs. The sequence is given above the spectrum and observed c and z-type fragment ions are indicated in the sequence with and , respectively. Doubly charged fragment ions of type c and z⋅ are labeled with +2, triply charged ions are of type c and z are indicated with *, z-type fragment ions that resulted from cleavage at cysteine with subsequent loss of the cysteine side chain are denoted in italic and charge reduced species are labeled in the spectrum with #. The spectrum was recorded at a resolution of 7500 at m/z 400 and all fragment ions have a mass accuracy of better than 5 ppm. Figure S3, RP-UHPLC chromatograms of Tv1 linear and oxidized peptide at 214nm. During a pilot folding reaction, over 90% of the linear Tv1 peptide fully oxidized and showed a peak at 1.58 minute in comparison to 1.83 minute linear Tv1 peak at the gradient of 0–75% buffer B (80% acetonitrile, 0.1% TFA) in buffer A (0.1% TFA) within two hours. Figure S4, Analysis of linear Tv1 peptide by MALDI-TOF mass spectrometry. MALDI-TOF spectrum of Tv1 peptide using α-Cyano-4-hydroxycinnamic acid matrix. Figure S5, Analysis of oxidized Tv1 peptide by MALDI-TOF mass spectrometry. MALDI-TOF spectrum of Tv1 peptide using α-Cyano-4-hydroxycinnamic acid matrix. Figure S6, RP-UHPLC analysis of partially reduced Tv1 peptide. On a UPLC (BEH 300 C18 1.7 μm, Waters Corporation, Milford, MA, USA) column, a linear gradient of 0–75% buffer B (80% acetonitrile, 0.1% TFA) in buffer A (0.1% TFA) over 6 minutes and peaks were assigned by their degree of reduction. Labels indicate how many disulfides are present. Figure S7, NOE contacts confirming the C7–C16 disulfide bond. An overlay of the HNHα fingerprint region of the NOESY (in black) and TOCSY (in blue) spectra shows NOE crosspeaks linking Cys 16 and Cys 7 as well as contacts in the residues flanking the C7–C16 disulfide bond (C7-S17, Y8-C16, G6-C16, G6-N18). Figure S8, Bundle of the 10 lowest energy structures of Tv1 after explicit water refinement. The 10 lowest energy structures are shown in stick representation, displaying the tight convergence found in the final structural bundle. Figure S9, Structure of Tv1. An overlay of a cartoon representation and a stick model of the lowest-energy structure of Tv1 shows the β-sheet character of the peptide and reveals the important role of the Tyr 8 side chain in the formation of a small hydrophobic core. Table S1, Predicted and observed of b and y ions of differentially alkylated peptides by auto and targeted MS/MS analysis. Table S2, Chemical shift assignments of Tv1 (in ppm). Table S3, Structural statistics for the final 10 models of Tv1. Table S4, Sample raw data of Tv1 bioactivity in polychaete worms. (DOC)