Electron Capture in Charge-Tagged Peptides. Evidence for the Role of Excited Electronic States

International audienceElectron capture dissociation (ECD) was studied with doubly charged dipeptide ions that were tagged with fixed-charge tris-(2,4,6-trimethoxyphenyl)phosphonium-methylenecarboxamido (TMPP-ac) groups. Dipeptides GK, KG, AK, KA, and GR were each selectively tagged with one TMPP-ac group at the N-terminal amino group while the other charge was introduced by protonation at the lysine or arginine side-chain groups to give (TMPP-ac-peptide + H)2+ ions by electrospray ionization. Doubly tagged peptide derivatives were also prepared from GK, KG, AK, and KA in which the fixed-charge TMPP-ac groups were attached to the N-terminal and lysine side-chain amino groups to give (TMPP-ac-peptide-ac-TMPP)2+ dications by electrospray. ECD of (TMPP-ac-peptide + H)2+ resulted in 72% to 84% conversion to singly charged dissociation products while no intact charge-reduced (TMPP-ac-dipeptide + H)+• ions were detected. The dissociations involved loss of H, formation of (TMPP + H)+, and N–C(alpha) bond cleavages giving TMPP-CH2CONH2+ (c0) and c1 fragments. In contrast, ECD of (TMPP-ac-peptide-ac-TMPP)2+ resulted in 31% to 40% conversion to dissociation products due to loss of neutral TMPP molecules and 2,4,6-trimethoxyphenyl radicals. No peptide backbone cleavages were observed for the doubly tagged peptide ions. Ab initio and density functional theory calculations for (Ph3P-ac-GK + H)2+ and (H3P-ac-GK + H)2+ analogs indicated that the doubly charged ions contained the lysine side-chain NH3+ group internally solvated by the COOH group. The distance between the charge-carrying phosphonium and ammonium atoms was calculated to be 13.1-13.2 Å in the most stable dication conformers. The intrinsic recombination energies of the TMPP+-ac and (GK + H)+ moieties, 2.7 and 3.15 eV, respectively, indicated that upon electron capture the ground electronic states of the (TMPP-ac-peptide + H)+• ions retained the charge in the TMPP group. Ground electronic state (TMPP-ac-GK + H)+• ions were calculated to spontaneously isomerize by lysine H-atom transfer to the COOH group to form dihydroxycarbinyl radical intermediates with the retention of the charged TMPP group. These can trigger cleavages of the adjacent N–C(alpha) bonds to give rise to the c1 fragment ions. However, the calculated transition-state energies for GK and GGK models suggested that the ground-state potential energy surface was not favorable for the formation of the abundant c0 fragment ions. This pointed to the involvement of excited electronic states according to the Utah-Washington mechanism of ECD

Interpretation of mass spectra

XVII+371hlm.;24c

Interpretation of Mass Spectra

xviii, 371 hal.; ill.; 25 c

Cascade dissociations of peptide cation-radicals. Part 1. Scope and effects of amino acid residues in penta-, nona-, and decapeptides.

International audienceAmino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS(3) spectra were elucidated for over 40 pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXX"AR and AAHAXAX"AR type, and AAHAAXX"AAR decapeptides. Peptide z(n) ions containing amino acid residues with readily transferrable benzylic or tertiary β-hydrogen atoms (Phe, Tyr, His, Trp, Val) underwent facile backbone cleavages to form dominant z(n-2) or z(n-3) ions. These backbone cleavages are thought to be triggered by a side-chain β-hydrogen atom transfer to the z ion C(α) radical site followed by homolytic dissociation of the adjacent C(α)-CO bond, forming x(n-2) cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable β-hydrogen atoms (Gly, Ala) do not undergo the z(n) → z(n-2) dissociations. The backbone cleavages compete with side-chain dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by α-hydrogen atom transfers that activate the C(β)-C(γ) or C(β)-heteroatom bonds for dissociations that dominate the MS(3) spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu, and Gln residues. The Lys, Arg, Gln, and Glu residues also participate in γ-hydrogen atom transfers that trigger other side-chain dissociations

Tunable charge tags for electron-based methods of peptide sequencing: design and applications.

International audienceCharge tags using basic auxiliary functional groups 6-aminoquinolinylcarboxamido, 4-aminopyrimidyl-1-methylcarboxamido, 2-aminobenzoimidazolyl-1-methylcarboxamido, and the fixed-charge 4-(dimethylamino)pyridyl-1-carboxamido moiety are evaluated as to their properties in electron transfer dissociation mass spectra of arginine C-terminated peptides. The neutral tags have proton affinities that are competitive with those of amino acid residues in peptides. Charge reduction by electron transfer from fluoranthene anion-radicals results in peptide backbone dissociations that improve sequence coverage by providing extensive series of N-terminal c-type fragments without impeding the formation of C-terminal z fragments. Comparison of ETD mass spectra of free and tagged peptides allows one to resolve ambiguities in fragment ion assignment through mass shifts of c ions. Simple chemical procedures are reported for N-terminal tagging of Arg-containing tryptic peptides