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

Author(s):

P. Boomathi Pandeswari, R. Nagarjuna Chary, A.S. Kamalanathan, Sripadi Prabhakar and Varatharajan Sabareesh*  

Abstract:


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: β-casein (bovine), β- 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.

Keywords:

Proteomics, Middle-down proteomics, Mass spectrometry, Electrospray Ionization, Arginine Modification, Tryptic peptides, 1, 2-Cyclohexanedione, Phenylglyoxal, Tandem mass spectrometry, Collision induced dissociation

Affiliation:

Centre for Bio-Separation Technology (CBST), Vellore Institute of Technology (VIT), Vellore, Tamil Nadu - 632014, Centre for Mass Spectrometry, Department of Analytical & Structural Chemistry, CSIR - Indian Institute of Chemical Technology (IICT), Hyderabad, Telangana - 500007, Centre for Bio-Separation Technology (CBST), Vellore Institute of Technology (VIT), Vellore, Tamil Nadu - 632014, Centre for Mass Spectrometry, Department of Analytical & Structural Chemistry, CSIR - Indian Institute of Chemical Technology (IICT), Hyderabad, Telangana - 500007, Centre for Bio-Separation Technology (CBST), Vellore Institute of Technology (VIT), Vellore, Tamil Nadu - 632014



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