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. ^

Process and outcome evaluation of the hepatitis B perinatal vaccination program in Houston, Texas, 1991--1993

A retrospective cohort study was designed to evaluate the compliance of vaccination dose schedules and vaccination effectiveness at 12 months of age among a total of 226 high-risk infants born to HBsAg-positive pregnant women who participated in the HBV Perinatal Vaccination Program in Houston, Texas, 1991-1993.^ The seroprevalence of HBsAg-positivity was 0.5% among pregnant women who attended prenatal clinics in Houston, Texas, 1991-1993. The Asian women had the highest seroprevalence rate (5.9%), followed by black (1.9%), white (0.7%), and Hispanic women (0.3%). The seroprevalence of HBsAg increased with age (p =.02); the highest seroprevalence rate found among the $>$40 group (5.4%), followed by the 20-40 age group, and the $<$20 age. A steady increase was observed in the number of infants, from 45 in 1991, to 103 in 1993. The majority of these infants were black (58.0%), followed by Hispanic (28.8%), Asian (8.4%), and white infants (4.0%). Significant increases were observed from 1991 to 1993 in the number of infants who initiated vaccination (86.7% to 98.1%, p =.02) and in those infants who were post-tested at 12 months of age (24.4% to 44.7%, p =.04). During the same period an increase was also observed in the number of infants who completed the vaccination dose schedules (62.2% to 72.8%, p =.37). The compliance rates were not statistically significant regarding gender, race or ethnicity, health service area, medical referral source, and residential geographic areas. About 56.0% of the reasons cited for non-compliance among the 144 infants who neither completed the vaccination dose schedules nor received the 12-month post-test were "moved," and "no response/not at home." A total of 82 infants completed the vaccination dose schedules and were post-tested at 12 months of age for anti-HBs-positivity, and 96.3% of these infants seroconverted. A race-specific statistically significant seroconversion difference was found among infants who received all vaccination doses and were post-tested at 12 months of age (100% for the black and the white, 96.3% for the Hispanic, and 80.0% for the Asians infants, p =.05).^ From a public health perspective, the HBV Perinatal Vaccination Program improved during its first three years (1991-1993). It was effective in preventing perinatal HBV infection in almost 97.0% of infants who were vaccinated and post-tested. To increase the efficiency and efficacy of the program, the following recommendations are proposed: (1) Increase the vaccination compliance rate by educating and improving the tracking, communication and coordination channels with those individuals involved in the process and by increasing staff resources. (2) Reduce the post-test vaccination non-compliance by post-testing infants simultaneously with third vaccination dose at 6 months of age, and only post-test those infants who are anti-HBs-negative at 9-12 months of age. (Abstract shortened by UMI.) ^