Title:Mimicking LysC Proteolysis by ‘Arginine Modification-cum-Trypsin
Digestion’: Comparison of Bottom-up & Middle-down Proteomic
Approaches by ESI Q-TOF MS
Volume: 28
Issue: 12
Author(s): P. Boomathi Pandeswari, R. Nagarjuna Chary, A.S. Kamalanathan, Sripadi Prabhakar and Varatharajan Sabareesh*
Affiliation:
- Centre for Bio-Separation Technology (CBST), Vellore Institute of Technology (VIT), Vellore, Tamil Nadu-632014, India
Keywords:
Proteomics, Middle-Down Proteomics, Mass Spectrometry, Electrospray Ionization, Arginine modification, Trypticpeptides, 1, 2-Cyclohexanedione, Phenylglyoxal, Tandem mass spectrometry, Collision Induced Dissociation.
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, and 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: The aim of this study is to obtain proteolytic peptides that resemble LysC peptides by using
'trypsin', which is a 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 of
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 demonstrated in this proof-of-concept study is not only useful to obtain
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 a 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.
