Mimicking LysC Proteolysis by ‘Arginine Modification-cum-Trypsin Digestion’: Comparison of Bottom-up & Middle-down Proteomic Approaches by ESI Q-TOF MS

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: -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.

Database Creator for Mass Analysis of Peptides and Proteins, DC-MAPP: A Standalone Tool for Simplifying Manual Analysis of Mass Spectral Data to Identify Peptide/Protein Sequences

“…These outputs were also verified manually. Similarly, the m / z values observed in the experimental MS and MS/MS data, which were recorded on the tryptic digest of CHD-modified proteins, were also queried as input to search in the respective database. Those CHD-modified peptides that matched with queried m / z values were obtained in the output, which were also confirmed manually (data not shown).…”

“…Eight standard proteins (see Table ) were chosen and subjected to two different proteolysis conditions: (i) standard trypsin digestion and (ii) trypsin digestion after arginine modification, viz., arginine modification-cum-trypsin digestion. 1,2-Cyclohexanedione (CHD) and phenylglyoxal (PG) were used for arginine modification of proteins, and further details regarding the experimental conditions of proteolysis have been already reported elsewhere . The mass spectral data were acquired on electrospray ionization quadrupole time-of-flight mass spectrometers (6540 and 6545 Ultra High Definition Accurate Mass Q-TOF LC/MS, Agilent Technologies) that were attached to a reverse phase ultra high-performance liquid chromatograph (1290 Infinity LC).…”

“…1,2-Cyclohexanedione (CHD) and phenylglyoxal (PG) were used for arginine modification of proteins, and further details regarding the experimental conditions of proteolysis have been already reported elsewhere. 21 The mass spectral data were acquired on electrospray Design and Description of DC-MAPP. Python version 3.7 was used for programming, and the graphical user interface (GUI) was built using tkinter.…”