Shyamalava Mazumdar

Electrospray Ionization Mass Spectrometry: A Technique to Access the Information beyond the Molecular Weight of the Analyte

The Electrospray Ionization (ESI) is a soft ionization technique extensively used for production of gas phase ions (without fragmentation) of thermally labile large supramolecules. In the present review we have described the development of Electrospray Ionization mass spectrometry (ESI-MS) during the last 25 years in the study of various properties of different types of biological molecules. There have been extensive studies on the mechanism of formation of charged gaseous species by the ESI. Several groups have investigated the origin and implications of the multiple charge states of proteins observed in the ESI-mass spectra of the proteins. The charged analytes produced by ESI can be fragmented by activating them in the gas-phase, and thus tandem mass spectrometry has been developed, which provides very important insights on the structural properties of the molecule. The review will highlight recent developments and emerging directions in this fascinating area of research.

Direct electrochemistry of heme proteins: effect of electrode surface modification by neutral surfactants

Direct electrochemical studies on horse heart myoglobin and horseradish peroxidase (HRP) have been carried out using tin-doped indium oxide (ITO) and surfactant modified glassy carbon working electrodes. These proteins show very slow electron transfer kinetics at metal or untreated electrodes. Moreover, small amounts of surface-active impurity were drastically affects the electrode reaction of these proteins. The results showed that modification of the electrode surface with neutral surfactants significantly improves the electrochemical response of myoglobin as well as of HRP. The electrode response was found to depend on the structure of the surfactants. The amount of surfactant required per unit area of the electrode surface to promote the maximum electron transfer rate constants was found to be constant. This indicated that the surfactant molecules interacted with the electrode surface in a specific manner and anchored the protein molecules to align in the suitable orientation. The hydrophobicity of the surfactants rather than their charge was found to be crucial in promoting the electrode response of these proteins at the glassy carbon electrode.

J- and H-Aggregates of Porphyrins with Surfactants: Fluorescence, Stopped Flow and Electron Microscopy Studies

The interaction of cationic surfactants such as CTAB (cetyl trimethyl ammonium bromide) with tetrakis-(4-sulfonato phenyl) porphine (H4TPPS2−), a dianion, leads to the formation of two premicellar aggregates of porphyrin when [CTAB] is below CMC (critical micelle concentration) and a micellized monomer when [CTAB] is above CMC. The premicellar aggregates are labeled as J- and H-aggregates because of their characteristic spectroscopic properties. Simple inorganic cations such as K+, Ba2+, Ca2+ and Zr4+ also induce the formation of the J-aggregate but not of the H-aggregate. The formation of the J-aggregate is preceded by kinetic intermediates, while no intermediate was observed prior to the formation of the H-aggregate. The rate of formation of the H-aggregate was faster than that of the J-aggregate. The fluorescence depolarization (anisotropy) properties of the monomer and the H- and J-aggregates were studied and compared. The size and structure of the J-aggregate were examined by transmission electron mi...

Direct correlation of the crystal structure of proteins with the maximum positive and negative charge states of gaseous protein ions produced by electrospray ionization

Electrospray mass spectrometric studies in native folded forms of several proteins in aqueous solution have been performed in the positive and negative ion modes. The mass spectra of the proteins show peaks corresponding to multiple charge states of the gaseous protein ions. The results have been analyzed using the known crystal structures of these proteins. Crystal structure analysis shows that among the surface exposed residues some are involved in hydrogen-bonding or salt-bridge interactions while some are free. The maximum positive charge state of the gaseous protein ions was directly related to the number of free surface exposed basic groups whereas the maximum negative charge state was related to the number of free surface exposed acidic groups of the proteins. The surface exposed basic groups, which are involved in hydrogen bonding, have lower propensity to contribute to the positive charge of the protein. Similarly, the surface exposed acidic groups involved in salt bridges have lower propensity to contribute to the negative charge of the protein. Analysis of the crystal structure to determine the maximum charge state of protein in the electrospray mass spectrum was also used to interpret the reported mass spectra of several proteins. The results show that both the positive and the negative ion mass spectra of the proteins could be interpreted by simple consideration of the crystal structure of the folded proteins. Moreover, unfolding of the protein was shown to increase the positive charge-state because of the availability of larger number of free basic groups at the surface of the unfolded protein.

Interaction of sodium dodecyl sulfate with human native and cross-linked hemoglobins: a transient kinetic study.

The interaction of sodium dodecyl sulfate (SDS) at a concentration range (0-515 microM) below the critical micelle concentration (CMC approximately 0.83 mM) with human native and cross-linked oxyhemoglobin (oxyHb) and methemoglobin (metHb) has been investigated by optical spectroscopy and stopped-flow transient kinetic measurements. It is observed that the interaction of SDS with human native and cross-linked oxyHb shows the disappearance of the bands of oxyHb at 541 and 576 nm and the appearance at 537 nm. The resultant spectra are characteristic of low spin (Fe(3+)) hemichrome. Similarly SDS has been found to convert human native and cross-linked high spin (Fe(3+)) metHb to low spin (Fe(3+)) hemichrome. The interaction of SDS with oxyHb suggests a conformational change of the protein in the heme pocket, which may induce the binding of distal histidine to iron leading to the formation of superoxide radical. The formation of hemichrome from metHb is found to be concentration-dependent with SDS. The stopped flow transient kinetic measurements of the interaction of SDS with metHb show that at least four molecules of SDS interact with one molecule of metHb. The interaction of SDS with human cross-linked oxy and met hemoglobin shows results similar to those for human native oxy and met hemoglobin indicating that the covalent modification does not alter the interaction of SDS with cross-linked hemoglobin.

Excited states of XH2+ (X=C, N, O, S) ions: a combined experimental and theoretical study

Excited electronic states of doubly charged molecular ions of the diatomic hydrides CH2+, NH2+, OH2+ and SH2+ are investigated by means of high-resolution translational energy spectrometry. Long-lived states of CH2+, NH2+ and SH2+ (but not OH2+) are observed. Potential energy functions are calculated using a semi-empirical molecular-orbital technique as well as a high-level ab initio method using large basis sets of at least double zeta plus polarisation quality. Translational energy spectra of singly charged hydride ions are also presented.

Evidence of Molecular Fragmentation inside the Charged Droplets Produced by Electrospray Process

The behavior of the analyte molecules inside the neutral core of the charged droplet produced by the electrospray (ES) process is not unambiguously known to date. We have identified interesting molecular transformations of two suitably chosen analytes inside the ES droplets. The highly stable Ni(II) complex of 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (1) that consists of a positive charge at the metal center, and the allyl pendant armed tertiary amine containing macrocycle 3,4,5:12,13,14-dipyridine-2,6,11,15-tetramethyl-1,7,10,16-tetraallyl-1,4,7,10,13,16-hexaazacyclooctadeca-3,13-diene (M4p) have been studied by ESI mass spectrometry as the model analytes. We have shown that these two molecules are not representatively transferred from solution to gas phase by ESI; rather, they undergo fragmentation inside the charged droplets. The results indicated that a charged analyte such as 1 was possibly unstable inside the neutral core of the ES droplet and undergoes fragmentation due to the Coulombic repulsion imparted by the surface protons. Brownian motion of the neutral analyte such as M4p inside the droplet, on the other hand, may lead to proton attachment on interaction with the charged surface causing destabilization that leads to fragmentation of M4p and release of resonance stabilized allyl cations from the core of the droplet. Detailed solvent dependence and collision-induced dissociation (CID) studies provided compelling evidences that the fragmentation of the analytes indeed occurs inside the charged ES droplets. A viable model of molecular transformations inside the ES droplet was proposed based on these results to rationalize the behavior of the analyte molecules inside the charged ES droplets.

Formation of doubly charged Co2+ ions: a combined experimental and theoretical study

The formation of doubly charged molecular ions of carbon monoxide is studied by means of ion translational energy spectrometry of products resulting from electron loss collisions of CO+ and He and double electron capture collisions of H+ and CO. Theoretical calculations of potential energy functions of several low-lying states of CO2+ have been carried out using a high level, all-electron ab initio technique using large Gaussian basis sets. Vertical double ionisation energies of CO are measured to be 40.21+or-0.35 eV and 39.45+or-0.20 eV. The former value pertains to a metastable CO2+ state whereas the latter is ascribed to a dissociative state. The energy difference of 0.76 eV is a measure of the splitting between the lowest 3 Pi and 1 Pi states.

Binding of camphor to Pseudomonas putida cytochrome P450cam: steady-state and picosecond time-resolved fluorescence studies

The binding of camphor to cytochrome P450cam has been investigated by steady‐state and time‐resolved tryptophan fluorescence spectroscopy to obtain information on the substrate access channel. The fluorescence quenching experiments show that some of the tryptophan residues undergo changes in their local environment on camphor binding. The time‐resolved fluorescence decay profile gives four lifetime components in the range from 99 ps to 4.5 ns. The shortest lifetime component assigned to W42 lies close to the proposed camphor access channel. The results show that the fluorescence of W42 is greatly affected on binding of camphor, and supports dynamic fluctuations involved in the passage of camphor through the access channel as proposed earlier on the basis of crystallographic, molecular dynamics simulation and site‐directed mutagenesis studies.

Non‐covalent dimers of the lysine containing protonated peptide ions in gaseous state: electrospray ionization mass spectrometric study

Study of the non-covalent molecular complexes in gas phase by electrospray ionization mass spectrometry (ESI-MS) represents a promising strategy to probe the intrinsic nature of these complexes. ESI-MS investigation of a series of synthetic octapeptides containing six alanine and two lysine residues differing only by their positions showed the formation of non-covalent dimers, which were preserved in the gas phase. Unlike the monomers, the dimers were found to show only singly protonated state. The decrease in the solvent polarity from water to alcohol showed enhanced propensity of formation of the dimer indicating that the electrostatic interaction plays a crucial role to stabilize the dimer. Selective functionalization studies showed that ε-NH(2) of lysine and C-terminal amide (-CONH(2)) facilitate the dimerization through intermolecular hydrogen bonding network.