Single cell analysis

The properties of organelles within a cell have been shown to be highly heterogeneous. Until now, it has been unclear just how much of this heterogeneity is endemic to the organelle subpopulations themselves and how much is actually due to stochastic cellular noise. An attractive approach for investigating the origins of heterogeneity among the organelles of a single cell is capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). As a proof of principle, we have used a single cell CE-LIF method to investigate the properties of endocytic (acidic) organelles and mitochondria. Our initial results suggests that the technique is capable of distinguishing subpopulations of these organelles.

Purification and analysis of subcellular compartments

Purification and analysis of subcellular compartments using microfluidic devices, capillary electrophoresis, free-flow electrophoresis, flow cytometry, and immunopurification

Direct analysis of tissue

The characterization of the chemical and biochemical properties of tissue biopsy specimens is commonplace in diagnoses, prognoses and biomedical research. Unfortunately, tissues are complex and heterogeneous structures that may contain different pathological processes within a few microns apart. More powerful diagnostic and prognostic tools could be possible if homogeneous sub-samples of tissue biopsies were to be analyzed by techniques that combine high separation power (e.g. capillary electrophoresis, CE) with a high yield of molecular information (e.g. mass spectrometry, MS), but these types of analysis are not yet readily available.
Our goal is to develop CE methodologies capable of directly sampling micron-sized regions of tissue cross-sections and analyzing the contents of such a region, either by laser-induced fluorescence detection (LIF) or tandem MS. As we develop these technologies, they are beign applied to investigate the metabolism and protein composition of human hepatocellular carcinoma (HCC) and non-HCC regions observed in the hepatic tissue of the same donor. If this plan is successful, this will be the first time that solid tissue samples of micrometer-size regions will be electrophoretically separated and then characterized by highly sensitive LIF or by information-rich MS.

Subcellular proteomics

Our current interests include:

1. proteomics of membrane proteins
2. identification of post-translational modification associated with aging

   Most current proteomic strategies have been developed to sequence and identify proteins based on their hydrophilic domains leaving the transmembrane domains of membrane domains underrepresented. We are investigating proteolysis in non-aqueous solvents as the means of  obtaining sequences from hydrophobic peptides from various subcellular membranes.
While post-translational modifications (e.g. carbonylation and nitrosylation) have been associated with aging, the temporal and tissue-specific distribution of such modifications is practically unknown. Furthermore, the effect of such modifications on cellular function has not been described. We are interested in micro-sampling tissue from a donor at several points in time and

Subcellular drug distribution and metabolism

Doxorubicin is an effective drug in the treatment of solid tumors as well as leukemia. However, the use of the drug results in toxic side effects such as cumulative cardiotoxicity. In order to increase the efficacy of this drug treatment or reduce toxic side effects, it is necessary to understand the fate of the drug after it enters into a cell. The current goals are to develop techniques to analyze the drug metabolism on a subcellular level and to examine different aspects, which might modify the drugs efficacy such as drug formulation (liposomal formulation and prodrugs) or the presence of p-glycoprotein.

Mitochondrial DNA analysis

The dynamics and organization of mitochondrial DNA (mtDNA) are still poorly understood. These processes are of utmost importance to mitochondrial function and to the etiology of mitochondrial-related diseases, particularly when coexisting wild type and mutated mtDNa are present within the same cell or tissue. Direct methods for quantitation of in individual mitochondria would be valuable tools to explore these processes. We are developing methods for determining mtDNA copy numbers in individual mitochondrial particles isolated by means of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. We are also investigating whether nucleoids (i.e. the unit of mitochondrial inheritance) can have both wild type and mutated mtDNA.

Detection of carbonylated proteins

Carbonyl formation in lipids, DNA, and proteins are considered markers of oxidative stress. We are interested in establishing correlations between the origins of carbonyl formation (i.e. production of reactive oxygen species) and the accumulation of carbonyl-modified biomolecules. In order to accomplish this goal we are developing sensitive methodologies to monitor attomole levels of reactive oxygen species and carbonyl contents in nanogram-mass tissue samples. In addition, we have collaborative proteomic projects aiming at the characterization and identification of carbonyl-modified proteins.

Superoxide release in mitochondria

Superoxide is the first reactive oxygen species (ROS) generated in mitochondria that may cause severe oxidative damage to mitochondrial DNA (mtDNA), lipids, and proteins upon transformation into other ROS. The resulting oxidative stress has been associated with aging and age-related diseases. Since superoxide is released asymmetrically on both sides of the mitochondrial inner membrane, forming two separate superoxide pools with different cellular effects, we are developing methods to simultaneously analyze superoxide released into the matrix and outside the mitochondria. We have implemented an MEKC-LIF method to separate and detect the accumulation of a superoxide-specific product of a recently developed probe, triphenylphosphonium-HE (TPP-HE), which is oxidized by superoxide. Initial results include simultaneous monitoring of superoxide production at either side of the mitochondrial inner membrane by complexes I or III, in samples taken from the 143B cell line and skeletal muscle of Fischer 344 rats. Future uses of this method include the comparison of inside/outside mitochondrial superoxide production in aging, and oxidative stress studies.