Mimicking LysC Proteolysis by ‘Arginine-Modification-cum-Trypsin digestion’: Comparison of Bottom-Up & Middle-Down Proteomic Approaches by ESI-QTOF-MS

Background: Middle-down (MD) proteomics is an emerging approach for reliable identification of post- translational modifications and isoforms, as this approach focuses on proteolytic peptides containing > 25 - 30 amino acid residues (a.a.r.), which are longer than typical tryptic peptides. Such longer peptides can be obtained by AspN, GluC, LysC proteases. Additionally, some special proteases were developed specifically to effect MD approach, e.g., OmpT, Sap9, etc. However, these proteases are expensive. Herein we report a cost-effective strategy, ‘arginine modification-cum trypsin digestion’, which can produce longer tryptic peptides resembling LysC peptides derived from proteins. Objective:: To obtain proteolytic peptides that resemble LysC peptides, by using 'trypsin', which is an less expensive protease. Methods: This strategy is based on the simple principle that trypsin cannot act at the C-termini of those arginines in proteins, whose sidechain guanidine groups are modified by 1,2-cyclohexanedione or phenylglyoxal. Results: As a proof of concept, we demonstrate this strategy on four models: -casein (bovine), - lactoglobulin (bovine), ovalbumin (chick) and transferrin (human), by electrospray ionization-mass spectrometry (ESI-MS) involving hybrid quadrupole time-of-flight. From the ESI-MS of these models, we obtained several arginine modified tryptic peptides, whose lengths are in the range, 30 - 60 a.a.r. The collision-induced dissociation MS/MS characteristics of some of the arginine modified longer tryptic peptides are compared with the unmodified standard tryptic peptides. Conclusion: The strategy followed in this proof-of-concept study, not only helps in obtaining longer tryptic peptides that mimic LysC proteolytic peptides, but also facilitates in enhancing the probability of missed cleavages by the trypsin. Hence, this method aids in evading the possibility of obtaining very short peptides that are < 5 - 10 a.a.r. Therefore, this is indeed an cost-effective alternative/substitute for LysC proteolysis and in turn, for those MD proteomic studies that utilize LysC. Additionally, this methodology can be fruitful for mass spectrometry based de novo protein and peptide sequencing.

Arginine modification of lycosin-I to improve inhibitory activity against cancer cells

Herein, arginine modification rendered Lycosin-I with higher anticancer activity, penetrability, and dissemination ability against solid tumor cells due to the optimized physicochemical properties and high serum stability.

Role of peptidylarginine deiminase 4 (PAD4) in pig parthenogenetic preimplantation embryonic development

SummaryArginine modification to citrulline (citrullination) is catalyzed by peptidylarginine deiminases (PADs) and one of the isomers PAD4 is shown to be involved in the gene regulation. In our previous paper we studied the localization and expression of PAD4 and the target of PAD4 in mammalian gametes and preimplantation embryos. In this study the role of PAD4 was examined in the pig diploid parthenogenetic preimplantation embryonic development. Knockdown of PAD4 by RNAi resulted in delayed development. Inhibition of PAD4 by a potent PAD4 inhibitor Cl-amidine from the time of activation for 24 h resulted in developmental arrest at the first cleavage. Inhibition at the later stages of development resulted in delayed or arrested development. A shorter exposure to Cl-amidine for 6 h at any stage of growth resulted in slow development. Thus, this study suggests that PAD4 activity is essential for the normal development of the embryos.

Arg-27, Arg-127 and Arg-155 in the β-trefoil protein barley α-amylase/subtilisin inhibitor are interface residues in the complex with barley α-amylase 2

Arginine residues in barley alpha-amylase/subtilisin inhibitor (BASI) involved in binding to barely alpha-amylase 2 (AMY2) were differentially labelled using AMY2 as protectant and phenylglyoxal (PGO) and [14C]PGO as modifying agents. Chymotryptic fragments of labelled BASI were purified by reverse-phase HPLC, and we concluded that the radiolabelled Arg-27, Arg-155 and most likely Arg-127, identified by amino acid, sequence and 14C analyses, are protected by AMY2. While Arg-106 and Arg-107 showed intermediate reactivity and apparently were only partly accessible, Arg-15, Arg-41 and Arg-61 reacted with PGO and were thus exposed in the BASI-AMY2 complex. Patterns of arginine modification by [14C]PGO in free or in AMY2-complexed BASI were consistent with the results of differential labelling. The AMY2-protected arginines in BASI are at a distance from each other, as deduced from crystal structures of different beta-trefoil proteins (Erythrina caffra and soybean trypsin inhibitors, interleukin-1 alpha and -1 beta and WASI, the wheat homologue), suggesting that the BASI-AMY2 complex has multiple contacts at a larger interface. Accordingly, 11-16-residue-long BASI oligopeptides synthesized to include Arg-27, Arg-106/Arg-107 or Arg-127 were unable to suppress the formation of BASI-AMY2 or the effect of an inhibitory monoclonal antibody to BASI. Since Arg-27 is not conserved in rice and wheat ASIs, we further propose that Arg-155 in BASI is the kinetically identified PGO-sensitive group that is essential for inhibition [Abe, Sidenius and Svensson (1993) Biochem. J. 293, 151-155].