Protein quaternary structure determination using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemical crosslinking

A means of analyzing protein quaternary structure using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI MS) and chemical crosslinking was evaluated. Proteins of known oligomeric structure, as well as monomeric proteins, were analyzed to evaluate the method. The quaternary structure of proteins of unknown or uncertain structure was investigated using this technique. The stoichiometry of recombinant E. coli carbamoyl phosphate synthetase and recombinant human farnesyl protein transferase were determined to be heterodimers using glutaraldehyde crosslinking, agreeing with the stoichiometry found for the wild type proteins. The stoichiometry of the gamma subunit of E. coli DNA polymerase III holoenzyme was determined in solution without the presence of other subunits to be a homotetramer using glutaraldehyde crosslinking and MALDI MS analysis. Chi and psi subunits of E. coli DNA polymerase III subunits appeared to form a heterodimer when crosslinked with heterobifunctional photoreactive crosslinkers.^ Comparison of relative % peak areas obtained from MALDI MS analysis of crosslinked proteins and densitometric scanning of silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels showed excellent qualitative agreement for the two techniques, but the quantitative analyses differed, sometimes significantly. This difference in quantitation could be due to SDS-PAGE conditions (differential staining, loss of sample) or to MALDI MS conditions (differences in ionization and/or detection). Investigation of pre-purified crosslinked monomers and dimers recombined in a specific ratio revealed the presence of mass discrimination in the MALDI MS process. The calculation of mass discrimination for two different MALDI time-of-flight instruments showed the loss of a factor of approximately 2.6 in relative peak area as the m/z value doubles over the m/z range from 30,000 to 145,000 daltons.^ Indirect symmetry was determined for tetramers using glutaraldehyde crosslinking with MALDI MS analysis. Mathematical modelling and simple graphing allowed the determination of the symmetry for several tetramers known to possess isologous D2 symmetry. These methods also distinguished tetramers that did not fit D2 symmetry such as apo-avidin. The gamma tetramer of E. coli DNA polymerase III appears to have isologous D2 symmetry. ^

Optimization of a DNSH passive sampling method to measure airborne carbonyls

Various airborne aldehydes and ketones (i.e., airborne carbonyls) present in outdoor, indoor, and personal air pose a risk to human health at present environmental concentrations. To date, there is no adequate, simple-to-use sampler for monitoring carbonyls at parts per billion concentrations in personal air. The Passive Aldehydes and Ketones Sampler (PAKS) originally developed for this purpose has been found to be unreliable in a number of relatively recent field studies. The PAKS method uses dansylhydrazine, DNSH, as the derivatization agent to produce aldehyde derivatives that are analyzed by HPLC with fluorescence detection. The reasons for the poor performance of the PAKS are not known but it is hypothesized that the chemical derivatization conditions and reaction kinetics combined with a relatively low sampling rate may play a role. This study evaluated the effect of absorption and emission wavelengths, pH of the DNSH coating solution, extraction solvent, and time post-extraction for the yield and stability of formaldehyde, acetaldehyde, and acrolein DNSH derivatives. The results suggest that the optimum conditions for the analysis of DNSHydrazones are the following. The excitation and emission wavelengths for HPLC analysis should be at 250nm and 500nm, respectively. The optimal pH of the coating solution appears to be pH 2 because it improves the formation of di-derivatized acrolein DNSHydrazones without affecting the response of the derivatives of the formaldehyde and acetaldehyde derivatives. Acetonitrile is the preferable extraction solvent while the optimal time to analyze the aldehyde derivatives is 72 hours post-extraction. ^