Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H(3)PO(4)), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H(3)PO(4) elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H(3)PO(4) upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13361-013-0770-2) contains supplementary material, which is available to authorized users

Biomimetic oxidation reactions of a naked manganese(V)-Oxo porphyrin complex

The intrinsic reactivity of a manganese(V)-oxo porphyrin complex, a typically fleeting intermediate in catalytic oxidation reactions in solution, has been elucidated in a study focused on its gas-phase ion-chemistry. The naked high-valent Mn(V)-oxo porphyrin intermediate 1 ([(tpfpp)Mn(V)O](+); tpfpp=meso-tetrakis(pentafluorophenyl) porphinato dianion), has been obtained by controlled treatment of [(tpfpp)Mn(III)]Cl (2-Cl) with iodosylbenzene in methanol, delivered in the gas phase by electrospray ionization and assayed by FT-ICR mass spectrometry. A direct kinetic study of the reaction with selected substrates, each containing a heteroatom X (X = S, N, P) including amines, sulfides, and phosphites, was thus performed. Ionic products arising from electron transfer (ET), hydride transfer (HT), oxygen-atom transfer (OAT), and formal addition (Add) may be observed, with a predominance of two-electron processes, whereas the product of hydrogen-atom transfer (HAT), [(tpfpp)Mn(IV)OH](+), is never detected. A thermochemical threshold for the formation of the product radical cation allows an evaluation of the electrontransfer ability of a Mn(V)-oxo complex, yielding a lower limit of 7.85 eV for the ionization energy of gaseous [(tpfpp)Mn(IV)O]. Linear free-energy analyses of the reactions of para-substituted N, N-dimethylanilines and thioanisoles indicate that a considerable amount of positive charge is developed on the heteroatom in the oxidation transition state. Substrates endowed with different heteroatoms, but similar ionization energy display a comparable reaction efficiency, consistent with a mechanism initiated by ET. For the first time, the kinetic acidity of putative hydroxo intermediates playing a role in catalytic oxidations, [(tpfpp)Fe(IV)OH](+) and [(tpfpp)Mn(IV)OH](+), has been investigated with selected reference bases, revealing a comparatively higher basicity for the ferryl, [(tpfpp)Fe(IV)O], with respect to the manganyl, [(tpfpp)Mn(IV)O], unit. Finally, the neat association reaction of 2 has been studied with various ligands showing that harder ligands are more strongly bound

Weather computing: l’indissolubile legame tra meteorologia e HPC

L’aspetto previsionistico meteorologico e quelli teorici e tecnologici della Computer Science sono, oggi, intimamente legati, sebbene la teoria di base che governa il comportamento e l’evoluzione dell’atmosfera sia stata sviluppata più di un centinaio di anni fa ovvero molto tempo prima che venisse costruito il primo computer elettronico. Accade molto spesso che i progressi compiuti nel settore tecnologico consentano di sviluppare, parametrizzare ed includere, nella descrizione modellistica dell’atmosfera, fenomenologie fisiche ritenute in un primo tempo ancillari rispetto alla descrizione di base, ma che, al crescere della raffinatezza della rappresentazione modellistica diventano fondamentali sia dal punto di vista qualitativo che quantitativo. È possibile tener conto del contributo di questi fenomeni solamente attraverso l’utilizzo massiccio di potenti supercalcolatori, a causa dell’enorme mole di calcoli necessari affinché essi siano adeguatamente “risolti” alla risoluzione spaziale e temporale necessaria, anche su scenari geografici relativamente poco estesi