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. ^

Development and testing of a procedure for systematically assigning gestational age for clinical and research use

A graphing method was developed and tested to estimate gestational ages pre-and postnatally in a consistent manner for epidemiological research and clinical purposes on feti/infants of women with few consistent prenatal estimators of gestational age. Each patient's available data was plotted on a single page graph to give a comprehensive overview of that patient. A hierarchical classification of gestational age determination was then applied in a systematic manner, and reasonable gestational age estimates were produced. The method was tested for validity and reliability on 50 women who had known dates for their last menstrual period or dates of conception, and multiple ultrasound examinations and other gestational age estimating measures. The feasibility of the procedure was then tested on 1223 low income women with few gestational age estimators. The graphing method proved to have high inter- and intrarater reliability. It was quick, easy to use, inexpensive, and did not require special equipment. The graphing method estimate of gestational age for each infant was tested against the last menstrual period gestational age estimate using paired t-Tests, F tests and the Kolmogorov-Smirnov test of similar populations, producing a 98 percent probability or better that the means and data populations were the same. Less than 5 percent of the infants' gestational ages were misclassified using the graphing method, much lower than the amount of misclassification produced by ultrasound or neonatal examination estimates. ^