TLC-Based Chemical Peptide Detection

The pre-purified peptide extract was chemically detected using Thin Layer Chromatography (TLC). A modified method previously described by [25 , 41 ] and also used by [24 (link)] was adopted for the TLC chemical detection. Pre-coated TLC plates (G₂₅₄ MERCK, Germany) were used. The solvent system used was n-butanol: acetic acid: water (3:1:1 v/v). The solvent reconstituted aqueous peptide fraction obtained from plant samples were spotted on the TLC plate and developed in the above-mentioned solvent system. The developed plates were allowed to dry, and sprayed with freshly prepared G-250 modified stain and Ninhydrin to detect the presence of circular and linear peptides, respectively. The sprayed plates were allowed to dry and viewed under UV at 245 nm and 365 nm. For MALDI TOF MS detection, 0.5 μL each of eluted fraction obtained from the C₁₈ pre-purification as described above was mixed well with 3 μL of CHCA (alpha cyano hydroxyl cinnamic acid) matrix, spotted on the MALDI target plate and dried away from light. Acquired spectra were processed using the 4800 Analyzer. The freeze dried fractions were reconstituted and again analysed for peptide mass peaks as described above [40 (link)].