Abstract: Structure and gas-phase fragmentation of tyrosyl-glycyl-tryptophan YGW^·+ and its derivative Ac-YGW-OMe^·+ have been studied using electrospray tandem mass spectrometry (ESI-MS/MS) combined with density functional theory (DFT). These peptide radical ions were generated via multistage collision-induced dissociation (CID) of transition metal-ligand-peptide tertiary complexes, Cu(L)M²⁺ (L=4′-chloro-2,2′:6′,2″-terpyridine (4Cl-tpy); M=YGW or Ac-YGW-OMe). Low-energy CID experiments revealed that the fragmentation mechanisms of YGW^·+ and Ac-YGW-OMe^·+ are dramatically different. YGW^·+ dissociate mainly through the loss of CO₂ and subsequent loss of indole radical to produce M-CO₂^·+(m/z 380.05) and M-CO₂-116⁺ (m/z 264.05) fragment ions. Other minor fragments include c₂+2H⁺ (m/z 238.04), G·GW⁺ (m/z 318.05) and 1H-indole·+ (m/z 117.23). In contrast, the main fragmentation of Ac-YGW-OMe^·+ is the loss of CH3OH to give rise to M-CH₃OH^·+ (m/z 448.14) product ion. Other minor fragments are z1-H·+ (m/z 200.93), c₂+2H⁺ (m/z 279.99) and M-CH₃COO·⁺ (m/z 421.16). The gas-phase fragmentation mechanisms of YGW^·+ and Ac-YGW-OMe·+ are proposed based on the aforementioned CID results. For YGW^·+, the major fragment ions M-CO₂^·+ (m/z 380.05) and M-CO₂-116⁺ (m/z 264.05) are generated through proton transfer from the carboxylic OH group to the amide oxygen to form carboxyl radical which undergoes cleavages of C_α-C and C_β-C_γ bonds, respectively. G·GW⁺ (m/z 318.05) fragment ion is formed by C_α-C_β bond cleavage with the loss of p-quinomethide, and c₂+2H⁺ (m/z 238.04) fragment ion is produced by proton transfer from the C_β-H of tryptophan to the amide oxygen to produce the β radical followed by N-C_α bond cleavage. For Ac-YGW-OMe^·+, the main product ion M-CH₃OH^·+ (m/z 448.14) is generated through proton transfer from three possible sources (C_α-H from glycine, Cα-H from tryptophan, C_β-H from side chain of tryptophan) to methyl ester oxygen followed by C-O bond cleavage, z₁-H^·+ (m/z 200.93) fragment ion is produced through proton transfer from the C_β-H of tryptophan to the amide oxygen to form β radical which undergoes N-C_α bond cleavage. The DFT calculation results suggest that the optimized geometries of YGW·+ and Ac-YGW-OMe^·+ are non-zwitterionic structures with a weak hydrogen bond between the amino nitrogen and the indole nitrogen (N₁-H⁺…N₄ (bond length ca. 2.043Å) in YGW^·+ and a strong hydrogen bond between the acetyl oxygen and the indole nitrogen (N4-H⁺…O₁ (bond length ca.1.666Å) in Ac-YGW-OMe^·+, respectively.