The selective covalent modification of singly protonated peptides in the gas-phase via ion/ion charge inversion reactions is demonstrated. Doubly deprotonated 4-formyl-1,3-benzene disulfonic acid serves as a reagent anion for forming a Schiff base via the reaction of a primary amine on the peptide and the aldehyde functionality of the reagent anion. The process is initiated by the formation of an ion/ion complex comprised to the two reactants. Ion trap collisional activation of the complex results in loss of water from the intermediate that gives rise to Schiff base formation. N-terminally acetylated peptides with no lysine residues do not undergo covalent bond formation upon reaction with the reagent anion. Rather, the adduct species simply loses the reagent either as a neutral species or as a deprotonated species. The ability to modify singly protonated peptide ions covalently and selectively opens up new possibilities for the analysis of peptides, and, possibly, other analyte species with primary amine functionalities.

Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, exist and in many cases are important for protein function. The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days. In this review we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples. Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of inter- and intra-molecular interactions in the global control of kinase function. Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signaling proteins. HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both classification of and greater understanding of the prevalence of protein motion.

Protonated tryptic peptides, somatostatin-14, and oxytocin have been subjected to reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic acid (FBDSA) in the gas phase. The major product is a negatively-charged complex comprised of the peptide and the reagent. Upon dehydration of the complex, all peptides show evidence for Schiff base formation involving a primary amine of the peptide. Some peptides also show evidence for the formation of a relatively strong electrostatic interaction without Schiff base formation (i.e., a mixture of isomeric precursor ions is generated upon dehydration of the complex). Ion trap collision-induced dissociation of the dehydration products from all peptides examined gave distinct product ion spectra relative to the deprotonated and protonated forms of the peptides. The distinct behavior of the modified ions is attributed to the highly stable charge carrying sulfonate group, which tends to inhibit intramolecular proton transfer in negatively charged species. Modified anions of the peptides with an intramolecular disulfide linkage show evidence for cleavage of both the disulfide linkage and an amide bond in the loop defined by the disulfide bond. Modification of protonated peptides via charge inversion with FBDSA is a useful means for generating novel and distinct ion-types that can provide complementary structural information upon subsequent activation to that obtained from dissociation of protonated or deprotonated forms of the peptide.

Reported in this contribution are the preparation and characterization of a series of Ru(2)(DMBA)(4) (DMBA = N,N'-dimethylbenzamidinate) bis(alkynyl) compounds, trans-Ru(2)(DMBA)(4)(X-gem-DEE)(2) [gem-DEE = σ-geminal-diethynylethene; X = H (1), Si(i)Pr(3) (2), Fc (3); 4-C(6)H(4)NO(2) (4), and 4-C(6)H(4)NMe(2) (5)]. Compounds 1-5 were characterized by spectroscopic and voltammetric techniques as well as the single-crystal X-ray diffraction studies of 2 and 3. Both the single-crystal structural data of compounds 2 and 3 and the spectroscopic/voltammetric data indicate that the gem-DEE ligands are similar to simple acetylides in their impact on the molecular and electronic structures of the Ru(2)(DMBA)(4) core. Furthermore, density functional theory calculations revealed more extensive π delocalization in aryl-donor-substituted gem-DEEs and that the hole-transfer mechanism will likely dominate the charge delocalization in Ru(2)-gem-DEE-based wires.

Presented herein is the synthesis and characterization of four diruthenium(II,III) compounds of formulas Ru2(Xap)4(CC-C6H4-4-NH2) (Xap is 2-anilinopyridinate, 1a; and 2-(3,5-dimethoxy)anilinopyridinate, 1b) and Ru2(Xap)4(CC-C6H4-3-NH2) (2a/2b). X-ray structural studies of compounds 1b and 2a revealed minimal changes in the coordination sphere of the Ru2 core. Voltammetric measurements showed that compounds 1 exhibit three one-electron redox processes: a reversible reduction of Ru2, a reversible oxidation of Ru2, and a quasi-reversible oxidation of an amino group. Compounds 2 display the same Ru2-based redox processes but not the −NH2 oxidation. Compounds 1a/1b were successfully converted to the corresponding diazonium salts [Ru2(Xap)4-(CC-C6H4-4-N2)](BF4) (3a/3b) via oxidation by nitrosonium tetrafluoroborate, which was generated in situ from t-BuONO and BF3. However, the attempt to convert compounds 2 to the corresponding diazonium salts was unsuccessful. DFT calculations of model compounds were performed to rationalize some unusual structural and electrochemical characteristics observed for compounds 1/2.