To understand the importance of arginine (Arg) in influencing electrospray ionization (ESI) - collision induced dissociation (CID) tandem mass spectrometry (MS/MS) behavior of peptides of lengths > ∼ 25 amino acid residues (a.a.r), we chose carbamidomethylated Insulin B-chain (30 a.a.r), glucagon (29 a.a.r) and melittin (26 a.a.r) as the models for this investigation. Also, two smaller peptides: Angiotensin II (8 a.a.r) and Bradykinin (9 a.a.r) were studied for better comprehension of the interplay between the influence of Arg and peptide's length. The motivation to study such longer peptides stems from middle-down proteomics, a recently emerging field encompassing investigations on proteolytic peptides longer than ∼25 a.a.r. CID MS/MS data of two different cases have been compared: (1) standard model peptides vs. chemically modified peptides, wherein sidechain guanidine group of Arg residues in these model peptides are selectively modified by 1,2-cyclohexanedione and phenylglyoxal; (2) standard model peptides vs. mutated model peptides, in which Arg residues in these model peptides are substituted by alanine (Ala) residues (Arg → Ala). Different types of stoichiometric products were obtained due to this chemical modification and each type of arginine-modified product was subjected to ESI CID MS/MS. The Arg → Ala mutated model peptides chosen for this study are: [R22A]-Insulin B-chain, [R17A & R18A]-Glucagon, [R22A & R24A]-Melittin and a shorter peptide: R2A-Angiotensin II. All experiments were performed in a quadrupole time-of-flight hybrid mass spectrometer, whereby CID (hexapole collision cell) was done by following fixed collision energy (CE) as well as ramped CE. Analysis of CID MS/MS spectra revealed that the sequence coverage of Arg → Ala mutated peptides was higher than the chemically modified peptides as well as their respective standard (unmodified) peptides. Examination of all the MS/MS data alluded that Arg has a greater influence on the CID MS/MS behavior of longer peptides, viz., lengths >25 a.a.r, than the smaller peptides. © 2020 Elsevier B.V.