No abstract

The results of time-resolved thermal dissociation measurements and molecular dynamic simulations are reported for gaseous deprotonated ions of the specific complexes of bovine beta-lactoglobulin (Lg) and a series of the fatty acids (FA): CH(3)(CH(2))(x)COOH, where x = 10, 12, 14, and 16. At the reaction temperatures investigated, 25-66 degrees C, the gaseous ions dissociate exclusively by the loss of neutral FA. According to the kinetic data, and confirmed by ion mobility measurements, the (Lg + FA)(7-) ions exist in two, noninterconverting structures designated the fast (Lg + FA)(f)(7-) and slow (Lg + FA)(s)(7-) components. The Arrhenius parameters for both components are sensitive to the length of the FA aliphatic chain. For the fast components, the activation energy (E(a)) increases in a nearly linear fashion, with each methylene group contributing approximately 0.8 kcal mol(-1) to E(a). This is similar to the contribution of -CH(2)- groups to the solvation of n-alkanes in nonpolar solvents. Furthermore, the magnitude of the E(a) values for the fast components is similar to the solvation enthalpies expected for the FA aliphatic chains in nonpolar and weakly polar solvents. The E(a) values determined for the slow components are larger than those of the fast components. Furthermore, the E(a) values do not vary in a simple fashion with the length of the aliphatic chain. Molecular dynamics simulations performed on the (Lg + PA) complex revealed that, depending on the charge configuration, the (Lg + PA)(7-) ion can exist in two distinct structures, which differ primarily by the position of the EF loop. In the open structure the EF loop is positioned away from the entrance to the hydrophobic cavity and the ligand is stabilized only through nonpolar intermolecular interactions. In the closed structure the EF loop covers the entrance of the cavity and the carboxylic group of PA participates in H-bonds with residues on the EF loop or residues located at the entrance of the cavity. The loss of ligand from the closed structure would require both the cleavage of the H-bonds and the nonpolar contacts. Taken together, these results suggest that the aliphatic chain of the FA remains bound within the hydrophobic cavity in the gas phase (Lg + FA)(7-) ions. Furthermore, the barrier to dissociation of the (Lg + FA)(f)(7-) ions reflects predominantly the cleavage of the nonpolar intermolecular interactions, while for the (Lg + FA)(s)(7-) ions the FA is stabilized by both nonpolar interactions and H-bonds.

Gas-phase ion/molecule chemistry has been combined with ion mobility separation and time-of-flight mass spectrometry to enable the characterization of large poly(ethylene glycol)s (PEGs) and PEGylated molecules (>40 kDa). A facile method is presented in which gas-phase superbases are reacted in the high-pressure source region of commercial TOF mass spectrometers to manipulate the charge states of large ions generated by electrospray ionization (ESI). Charge stripping decreases the spectral congestion typically observed in ESI mass spectra of high molecular weight polydisperse PEGylated molecules. From these data, accurate average molecular weights and molecular weight distributions for synthetic polymers and PEGylated proteins are determined. The average MW measured for PEGylated Granulocyte colony-stimulating factor (rh-GCSF, 40 726.2 Da) is in good agreement with the theoretical value, and a 16 Da mass shift is easily observed in the spectrum of an oxidized form of the heterogeneous PEGylated protein. Ion mobility separations can fractionate PEGs of different chain length; when coupled with charge stripping ion/molecule reactions, ion mobility mass spectrometry (IMMS) offers several analytical advantages over mass spectrometry alone for the characterization of large PEGylated molecules including enhanced dynamic range, increased sensitivity, and specificity. Low abundance free PEG in a PEGylated peptide preparation, which is not directly detectable by mass spectrometry, can be easily observed and accurately quantified with gas-phase ion/molecule chemistry combined with ion mobility mass spectrometry.

Over the past two decades orthogonal acceleration time-of-flight has been the de facto analyzer of choice for solution and membrane soluble protein native mass spectrometry (MS) studies; this however is gradually changing. Here we compare three MS instruments, the Q-ToF, the Orbitrap and the FT-ICR to analyze, under native instrument and buffer conditions, the 7-transmembrane helical protein bacteriorhodopsin-octylglucoside micelle complex and the empty nanodisc (MSP1D1-Nd) using both MS and tandem-MS modes of operation. Bacteriorhodopsin can be released from the octylglucoside-micelle efficiently on all three instruments (MS-mode of operation) producing a narrow charge state distribution (z = 8+ to 10+) by either increasing the source lens or collision cell (or HCD) voltages. A lower center-of-mass collision energy (0.20–0.41 eV) is required for optimal bacteriorhodopsin liberation on the FT-ICR, in comparison to the Q-ToF and Orbitrap instruments (0.29–2.47 eV). The empty MSP1D1-Nd can be measured with relative ease on a three instruments, resulting in a highly complex spectrum of overlapping, polydisperse charge state; a consequence of varying levels of phospholipid incorporation. There is a measurable difference in MSP1D1-Nd charge state distribution (z = 15+ to 26+), average molecular weight (141.7 to 169.6 kDa) and phospholipid incorporation number (143 to 184) under low activation conditions. Utilizing tandem-MS, bacteriorhodopsin can be effectively liberated from the octylglucoside-micelle by collisional (Q-ToF and FT-ICR) or continuous IRMPD activation (FT-ICR). MSP1D1-Nd spectral complexity can also be significantly reduced by tandem-MS (Q-ToF and FT-ICR) followed by mild collisional or continuous IRMPD activation, resulting in a spectrum in which the charge state and phospholipid incorporation levels can easily be determined.