The most successful approach to characterize subcellular proteins still requires a 2D-gel electrophoresis separation of the proteins of interest followed protein extraction, most times protein digestion, and then mass spectrometric characterization. However, most expert would agree that there are three stumbling steps in the analysis of these proteomes. First, a lot of the organelle proteins are hydrophobic because they are found embedded in membranes. Their hydrophobicity affects their solubility which compromises their extraction and further separation on a gel system. Second, a protein fraction usually is contaminated by those proteins from other subcellular compartments or the cytoplasm even when using the most sophisticated purification strategies. Third, the methodology for characterization of proteomes is lengthy and unable to characterize complete proteomes.

Direct MALDI-TOF MS of mitochondria

Ideally emerging proteomic strategies for organelle proteins need to address these problems. Instead of attempting to extract proteins from a given tissue or subcellular fraction, proteins still bound to an organelle may be handled by manipulating and separating the whole organelle in lieu of protein extraction, purification, and 2D gel separation.

We are investigating how to determine proteome profiles or specific proteins in intact liposomes, mitochondria, nuclei and platelet derived microparticles after they are electrophoretically manipulated. Because the separation conditions are adjustable so that the migration time becomes a function of microparticle size or zeta potential (i.e. surface electrical charge), it is envisioned that, by using electrophoretic properties, subpopulations of a given organelle that vary in surface phospholipid composition, protein expression, or abundance of sialic acid may be sorted and then analyzed by matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS). Similarly, the subcellular distribution of fluorescent proteins such as fusion product of synthetases with green fluorescent protein (GFP) may be investigated.

Upon a development of these new proteomic strategies we plan to use them to (1) link mitochondrial proteome with the age of the donor cell or tissue and to (2) link platelet derived microparticles with disease status. This project is a collaboration with Nigel Key, M.D., Medical School, University of Minnesota. (3). Explore the subcellular distribution of fusion proteins composed of aminoacyl-tRNA synthetases and GFP. This project is a collaboration with Professor Karin Musier-Forsyth, Department of Chemistry, University of Minnesota.

Participants