Final Technical Report
Two primary technologies have been employed for analysis and measurement of the Synechocystis proteome. (1) 2D-gel electrophoresis. Currently one of the most reliable options in quantitative proteomics, typical 2D-gel experiments use isoelectric focusing (IEF) in the first dimension. In the case of membrane proteins, detergents must be added to maintain their solubility though only neutral/zwitterionic surfactants are compatible with the IEF process. We have optimized 2D gel separations for Synechocystis proteins extracted and separated into soluble and membrane subfractions. The resolution and coverage of integral membrane proteins is only marginally satisfactory and alternatives to the first dimension are being considered. Size-exclusion chromatography under non-denaturing conditions was one option that was explored but resolution was insufficient for subfractionation of the membrane-bound proteome. A more highly resolving technique, the ''Blue-native gel'' has proven excellent for Synechocystis and we plan to set up this technology in the near future. Proteins with altered expression are being identified through standard LCMSMS technologies. The analysis of PSI, PSII and SDH deficient mutants is completed, establishing the comparative aspect of the project for integration with the ultrastructural and metabolomic experiments at ASU. We are also looking forward to receiving ftsZ and VIPP1 interruption mutants to explore the effects on the proteome of cell enlargement and disruption of thylakoid biogenesis, respectively. (2) 2D liquid chromatography with mass spectrometry of intact proteins. Early experiments with total membrane protein extracts of Synechocystis showed that the spatial resolution of the reverse-phase separation used in front of the mass spectrometer limited detection to the one hundred or so most abundant proteins. The intact mass tags (IMTs) measured in this experiment represent the first of these measurements that will ultimately define the entire proteome. While some of the IMTs were matched to masses calculated from translations of genomic open-reading frames allowing reasonably confident identification of about half of them (hypothetical IMTs), we are currently validating identifications using a combination of peptide mass fingerprinting after cyanogen bromide cleavage and LC-MSMS after trypsin, of protein in fractions collected during LC-MS+. In order to gain more complete proteome coverage we are applying a liquid separation in front of the LC-MS+ experiment. Size-exclusion chromatography is the first separation technology to be employed, yielding immediate benefits, while still not satisfactory for overall resolution of complexes. Total membranes were solubilized with dodecyl maltoside (1.5%) and separated on deactivated silica (G 4000 SW). LC-MS+ analysis of less-retained chlorophyll-containing fractions, using reverse-phase and size-exclusion technologies, yielded intact protein mass spectra of the two large photosystem I subunits PsaA and PsaB as well as many other IMTs (Figures 1 & 2). These integral membrane proteins have eleven transmembrane helices and, at 81 and 83 kDa, represented one of the most significant challenges to the intact protein molecular weight approach. The identities of the proteins were confirmed by peptide mass fingerprinting and while there is good general agreement between measured and calculated masses it is noted that modest post-translational modifications are necessary to account for the measured molecular weights of the intact proteins. Whether these discrepancies are due to genuine post-translational modifications or DNA sequence errors remains to be determined. The data have been published allowing us to claim to be the first to have completed high-resolution electrospray-ionization mass spectrometry of the core subunits of Photosystem II, Photosystem I and the cytochrome b{sub 6}f complex providing effective proof-of-principle for application of the intact mass approach to the integral membrane proteome. Significantly, we reported greater integral membrane proteome coverage than a colleague studying thylakoids of Arabidopsis illustrating the benefits of the technique over sequential organic extraction of membrane proteins and 1D-gel analysis. The homogeneity of the PsaA and PsaB protein mass spectra attest to the quality of material grown at ASU and the viability of extraction and work up of the material after transport to UCLA.
- Research Organization:
- Univ. of California, Los Angeles, CA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG03-01ER15253
- OSTI ID:
- 842077
- Report Number(s):
- DOE/ER/15253; TRN: US200707%%170
- Country of Publication:
- United States
- Language:
- English
Similar Records
Proteomic Analyses using High-Efficiency Separations and Accurate Mass Measurements
Discovery top-down proteomics in symbiotic soybean root nodules