Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Chemical modification of proteins to mimic LysC proteolysis: Application of 1,2-dicarbonyl compounds for arginine modification (#764)

Boomathi P Pandi 1 , Nagarjuna R Chary 2 , A.S. Kamalanathan 1 , Sripadi Prabhakar 2 , Varatharajan Sabareesh 1
  1. Advanced Centre for Bio Separation Technology (CBST), Vellore Institute of Technology (VIT), Vellore - 632014, Tamil Nadu, India
  2. Centre for Mass Spectrometry, Analytical Department, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, Telangana - 500007, India


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.

  1. Takahashi, K., (1968). The Reaction of phenylglyoxal with arginine residues in proteins. J. Biol. Chem. 243, 6171-6179
  2. Patthy, L., and Smith, E.L. (1975). Reversible modification of arginine residues. J. Biol. Chem. 250, 557-564
  3. Brock, J.W.C., Cotham, W.E., Thorpe, S.R., Baynes,J.W., and Ames,J.M. (2007). Detection and Identification of arginine modifications on methylglyoxal-modified ribonuclease by mass spectrometric analysis. J.Mass Spectrom. 42, 89-100