Karen J. Light-Wahl

Observation and Implications of High Mass-to-Charge Ratio Ions from Electrospray Ionization Mass Spectrometry

High mass-to-charge ratio ions (> 4000) from electrospray ionization (ESI) have been observed for several proteins, including bovine cytochrome c (Mr 12,231) and porcine pepsin (Mr 34,584), by using a quadrupole mass spectrometer with an m/z 45,000 range. The ESI mass spectrum for cytochrome c in an aqueous solution gives a charge state distribution that ranges from 12 + to 2 +, with a broad, low-intensity peak in the mass-to-charge ratio region corresponding to the [M + H]+ ion. the negative ion ESI mass spectrum for pepsin in 1% acetic acid solution shows a charge state distribution ranging from 7− to 2−. To observe the [M - H]− ion, harsher desolvation and interface conditions were required. Also observed was the abundant aggregation of the protens with average charge states substantially lower than observed for their monomeric counterparts. The negative ion ESI mass spectrum for cytochrome c in 1–100 mM NH4OAc solutions showed greater relative abundances for the higher mass-to-charge ratio ions than in acuidic solutions, with an [M - H]− ion relative abundance approximately 50% that of the most abundant charge state peak. The observation that protein aggregates are formed with charge states comparable to monomeric species (at fower mass-to-charge ratios) suggests that the high mass-to-charge ratio monomers may be formed by the dissociation of aggregate species. The observation of low charge state and aggregate molecular ions concurrently with highly charged species may serve to support a variation of the charged residue model, originally described by Dole and co-workers (Dole, M., et al. J. Chem. Phys.1968, 49, 2240; Mack, L. L., et al. J. Chem. Phys.1970, 52, 4977) which involves the Coulombically driven formation of either very highly solvated molecular ions or lower ananometer-diameter droplets.

Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H]15+ ion being the most intense peak. With the addition of H2O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.

Observation of noncovalent complexes to the avidin tetramer by electrospray ionization mass spectrometry.

Intact avidin-biotin and avidin-biotin maleimide noncovalent complexes have been observed by electrospray ionization mass spectrometry (ESI-MS) by using an extended mass range quadrupole mass spectrometer. By utilizing mild ES1 interface conditions, the expected solution behavior of four biotin or biotin maleimide molecules noncovalently binding to each avidin tetramer can be preserved in the gas phase. The ESI-MS results show the appropriate mass additions of 973 ± 60 Da for biotin and 1802 ± 40 Da for biotin maleimide to the avidin tetramer species. These results support the hypothesis that substantial retention of higher order structure is possible in the gas phase by using gentle ESI conditions.

New developments in microscale separations and mass spectrometry for biomonitoring: Capillary electrophoresis and electrospray ionization mass spectrometry

In this article, we briefly highlight the use of capillary electrophoresis for sampling, manipulating, and separating extremely small sample sizes. The extraordinary sensitivity that can be obtained by combined capillary electrophoresis-mass spectrometry is then demonstrated using recent results. We briefly describe the ability to detect noncovalently associated complexes (e.g., double-stranded DNA) by electrospray ionization-mass spectrometry, and conclude with recent results that show the potential for using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry for characterization of biomolecules.

A uniquely modified RNA: Introduction of a single RNA cleavage agent into the M1 ribozyme

Abstract We describe an efficient, four-step synthesis of an N 4 -modified cytidine phosphoramidite reagent ( 1 ) that permits nonnative functionality to be introduced into a synthetic oligoribonucleotide. This reagent was used to prepare a 377-nt E. coli M1 ribozyme equipped with a single RNA cleavage agent at nucleotide 11. We describe an efficient, four-step synthesis of an N 4 -modified cytidine phosphoramidite reagent ( 1 ) that permits nonnative functionality to be introduced into a synthetic oligoribonucleotide. This reagent was used to prepare a 377-nt E. coli M1 ribozyme equipped with a single RNA cleavage agent at nucleotide 11.