Protein Identification via MALDI-TOF MS

MALDI-TOF was employed to identify the proteins which showed reactivity. In-gel, digestions of spots were done according to the protocol reported by Shevchenko et al. (1996) (link). The procedure was adopted from our earlier study (Singh et al., 2015 (link), 2018 (link)). In brief, an automated robotic enzymatic (Trypsin) digestion of spots of interest was done by using a protein digester (Model Investigator ProPrep, Genomic Solutions Ltd., UK) and was further purified by using ZipTipC18 (Millipore). The purified proteins were then applied to AnchorChip (Bruker) with 2 μl of the matrix [a saturated solution of α-cyano-4-hydroxycinnamic acid (HCCA) made in 50% ACN and 0.2% TFA]. Mass spectra of digested peptides were acquired by Autoflex II TOF/TOF50 (Bruker Daltonik GmgH, Leipzig, Germany) in positive reflectron mode and the detection range of 500–3,000 m/z. The peptide mass fingerprints were searched by using the Mascot Wizard program (Matrix Science, Ltd., London, United Kingdom). Search parameters used in MS/MS for the identification of spots were peptide mass tolerance ±0.5 Da ppm, peptide charge state 1+, and maximum missed cleavages one.