PARENT SESSION
Posters P8B Supermolecular organization of the photosynthetic apparatus. Abstracts (592-611)

Intact protein mass measurements in proteomics: A workflow for photosynthetic membranes. Julian Whitelegge^*,1, Puneet Souda¹, Stephen Gómez¹, Alek Dooley¹, Kym Faull¹, Dmitrii Vavilin², Willem Vermaas², ¹ The Pasarow Mass Spectrometry Laboratory, Los Angeles, CA, usa² School of Life Sciences, Tempe, Arizona, usa

ABSTRACT- Proteomics seeks to monitor the flux of protein through cells under variable developmental and environmental influences as programmed by the genome. Consequently, it is necessary to measure changes in protein abundance and turnover rate as faithfully as possible. In the absence of non-invasive technologies, the majority of proteomics approaches involve destructive sampling at various time points to obtain snapshots that periodically report the genomes product. The work has fallen to separations technologies, coupled to mass spectrometry for high-throughput protein identification. Quantitation has become the major challenge facing proteomics as the field matures. A common feature of quantitative proteomics is the use of stable isotope coding to distinguish control and experimental samples in a mixture that are profiled in a single experiment. In vivo pulse-chase with stable isotopes allow for measurement of protein turnover rates. Protocols for recording mass spectra of intact integral membrane proteins from thylakoid and other membranes have been established. Liquid chromatography in a variety of aqueous/organic solvent mixtures has been interfaced with electrospray ionization providing well-resolved mass spectra of polytopic membrane proteins. The approach has been extended to membrane protein complexes as well as sub-domains and, by incorporating a split solvent line, protein fractions are available for cleavage and tandem mass spectrometry (LCMS+). The complexity of whole membrane systems demands separations in two or more dimensions. Interface of 2D-gel electrophoresis with intact protein mass spectrometry is inconvenient and 2D-chromatography systems are being evaluated against the separation power of 2D-gels. A rational workflow for proteomics of photosynthetic membranes that allows for intact protein mass measurements and localization of post-translational modifications as well as an alternative strategy for quantitative proteomics is presented.

KEY WORDS: proteomics, liquid chromatography, mass spectrometry, post-translational modifications