We are interested to develop a new method for an emerging area, middle-down proteomics (MDP). In MD approach, proteases such as LysC, AspN, etc. are used to produce longer proteolytic peptides, which yields better sequence coverage than bottom-up approach and hence, post-translational modifications can be detected more reliably (e.g., in histones and antibodies). We apply the strategy of modifying the guanidine side chain of arginines of proteins by 1,2-cyclohexanedione (CHD) or phenylglyoxal (PG), prior to trypsin digestion, which would result in ‘longer tryptic peptides’ and such arginine-modified tryptic peptides mimic LysC derived peptides.
Before applying this method for proteomics, we investigated five model proteins: β-lactoglobulin, β-casein, RNase A, ovalbumin and human transferrin. Carbamidomethylation of proteins was done before arginine modification and ~100 molar excess of CHD or PG was used (16 hrs, pH 8.4 (borate), ~250 C) for arginine modification reactions. Subsequently, each sample was digested with trypsin (370C) for different incubation times, which was monitored by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS: 1290 Infinity LC attached to 6540 or 6545 Q-TOF (Agilent) and Acquity UPLC attached with Quattro Premier XE (Waters)).
Three arginine-modified tryptic peptides of lengths in the range: ~26 - 50 amino acid residues (a.a.r), were detected from each of β-lactoglobulin and β-casein. In all these modified tryptic peptides, one molecule of CHD or PG was covalently added to the sidechain of the arginine residue. Tryptic peptides of very short lengths (< 5 a.a.r) and not longer than 25 a.a.r. were not observed from arginine-modified RNase A. Similarly, longer arginine-modified tryptic peptides were observed in other model proteins as well.
Thus, in cases, where LysC is useful for MD approach based proteomics and protein sequencing, our strategy of arginine-modification-cum-trypsin digestion can be a new approach, which can be an alternative method and cost-effective too.