Nilesh S. Tannu

Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling.

Quantitative proteomics is the workhorse of the modern proteomics initiative. The gel-based and MuDPIT approaches have facilitated vital advances in the measurement of protein expression alterations in normal and disease phenotypic states. The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the multiplexing fluorescent two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). 2D-DIGE is based on direct labeling of lysine groups on proteins with cyanine CyDye DIGE Fluor minimal dyes before isoelectric focusing, enabling the labeling of 2–3 samples with different dyes and electrophoresis of all the samples on the same 2D gel. This capability minimizes spot pattern variability and the number of gels in an experiment while providing simple, accurate and reproducible spot matching. This protocol can be completed in 3–5 weeks depending on the sample size of the experiment and the level of expertise of the investigator.

Comparative Proteomes of the Proliferating C2C12 Myoblasts and Fully Differentiated Myotubes Reveal the Complexity of the Skeletal Muscle Differentiation Program

When cultured in low serum-containing growth medium, the mouse C2C12 cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C2C12 cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C2C12 myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C2C12 myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C2C12 myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation.

Methods for proteomics in neuroscience

Proteomics reveals complex protein expression, function, interactions and localization in different phenotypes of neuron. As proteomics, regarded as a highly complex screening technology, moves from a theoretical approach to practical reality, neuroscientists have to determine the most-appropriate applications for this technology. Even though proteomics compliments genomics, it is in sheer contrast to the basically constant genome due to its dynamic nature. Neuroscientists have to surmount difficulties particular to the research in neuroscience; such as limited sample amounts, heterogeneous cellular compositions in samples and the fact that many proteins of interest are hydrophobic proteins. The necessity of exclusive technology, sophisticated software and skilled manpower tops the challenge. This review examines subcellular organelle isolation, protein fractionation and separation using two-dimensional gel electrophoresis (2-DGE) as well as multi-dimensional liquid chromatography (LC) followed by mass spectrometry (MS). The methods for quantifying relative gene product expression between samples (e.g., two-dimensional difference in gel electrophoresis (2D-DIGE), isotope-coded affinity tag (ICAT) and iTRAQ) are elaborated. An overview of the techniques used currently to assign post-translational modification status on a proteomics scale is also evaluated. The feasible coverage of the proteome, ability to detect unique cell components such as post-synaptic densities and membrane proteins, resource requirements and quantitative as well as qualitative reliability of different approaches is also discussed. While there are many challenges in neuroproteomics, this field promises many returns in the future.

Cytosolic proteomic alterations in the nucleus accumbens of cocaine overdose victims

Chronic cocaine use in humans and animal models is known to lead to pronounced alterations in neuronal function in the nucleus accumbens (NAc), a brain region associated with drug reinforcement. Two-dimensional gel electrophoresis was used to compare protein alterations in the NAc between cocaine overdose (COD) victims (n=10) and controls (n=10). Following image normalization, spots with significantly differential image intensities (P<0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser desorption ionization-time of flight-time of flight. A total of 1407 spots were found to be present in a minimum of five subjects per group and the intensity of 18 spots was found to be differentially abundant between the groups, leading to positive identification of 15 proteins by peptide mass fingerprinting (PMF). Of an additional 37 protein spots that were constitutively expressed, 32 proteins were positively identified by PMF. Increased proteins in COD included β-tubulin, liprin-α3 and neuronal enolase, whereas decreased proteins included parvalbumin, ATP synthase β-chain and peroxiredoxin 2. The present data provide a preliminary protein profile of COD, suggesting the involvement of novel proteins and pathways in the expression of this complex disease. Additional studies are warranted to further characterize alterations in the differentially regulated proteins. Understanding the coordinated involvement of multiple proteins in cocaine abuse provides insight into the molecular basis of the disease and offers new targets for pharmacotherapeutic intervention for drug abuse-related disorders.

Integrative proteomic analysis of the nucleus accumbens in rhesus monkeys following cocaine self-administration

The reinforcing effects and long-term consequences of cocaine self-administration have been associated with brain regions of the mesolimbic dopamine pathway, namely the nucleus accumbens (NAc). Studies of cocaine-induced biochemical adaptations in rodent models have advanced our knowledge; however, unbiased detailed assessments of intracellular alterations in the primate brain are scarce, yet essential, to develop a comprehensive understanding of cocaine addiction. To this end, two-dimensional difference in gel electrophoresis (2D-DIGE) was used to compare changes in cytosolic protein abundance in the NAc between rhesus monkeys self-administering cocaine and controls. Following image normalization, spots with significantly differential image intensities (P<0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser-desorption ionization time-of-flight time-of-flight (MALDI-TOF-TOF). In total, 1098 spots were subjected to statistical analysis with 22 spots found to be differentially abundant of which 18 proteins were positively identified by mass spectrometry. In addition, approximately 1000 protein spots were constitutively expressed of which 21 proteins were positively identified by mass spectrometry. Increased levels of proteins in the cocaine-exposed monkeys include glial fibrillary acidic protein, syntaxin-binding protein 3, protein kinase C isoform, adenylate kinase isoenzyme 5 and mitochondrial-related proteins, whereas decreased levels of proteins included β-soluble N-ethylmaleimide-sensitive factor attachment protein and neural and non-neural enolase. Using a complimentary proteomics approach, the differential expression of phosphorylated proteins in the cytosolic fraction of these subjects was examined. Two-dimensional gel electrophoresis (2DGE) was followed by gel staining with Pro-Q Diamond phosphoprotein gel stain, enabling differentiation of approximately 150 phosphoprotein spots between the groups. Following excision and trypsin digestions, MALDI-TOF-TOF was used to confirm the identity of 15 cocaine-altered phosphoproteins. Significant increased levels were detected for γ-aminobutyric acid type A receptor-associated protein 1, 14-3-3 γ-protein, glutathione S-transferase and brain-type aldolase, whereas significant decreases were observed for β-actin, Rab GDP-dissociation inhibitor, guanine deaminase, peroxiredoxin 2 isoform b and several mitochondrial proteins. Results from these studies indicate coordinated dysregulation of proteins related to cell structure, signaling, metabolism and mitochondrial function. These data extend and compliment previous studies of cocaine-induced biochemical alterations in human postmortem brain tissue, using an animal model that closely recapitulates the human condition and provide new insight into the molecular basis of the disease and potential targets for pharmacotherapeutic intervention.

Biomarkers for the Development of New Medications for Cocaine Dependence

There has been significant progress in personalized drug development. In large part, this has taken place in the oncology field and been due to the ability of researchers/clinicians to discover and develop novel drug development tools (DDTs), such as biomarkers. In cancer treatment research, biomarkers have permitted a more accurate pathophysiological characterization of an individual patient, and have enabled practitioners to target mechanistically the right drug, to the right patient, at the right time. Similar to cancer, patients with substance use disorders (SUDs) present clinically with heterogeneous symptomatology and respond variably to therapeutic interventions. If comparable biomarkers could be identified and developed for SUDs, significant diagnostic and therapeutic advances could be made. In this review, we highlight current opportunities and difficulties pertaining to the identification and development of biomarkers for SUDs. We focus on cocaine dependence as an example. Putative diagnostic, pharmacodynamic (PD), and predictive biomarkers for cocaine dependence are discussed across a range of methodological approaches. A possible cocaine-dependent clinical outcome assessment (COA)—another type of defined DDT—is also discussed. At present, biomarkers for cocaine dependence are in their infancy. Much additional research will be needed to identify, validate, and qualify these putative tools prior to their potential use for medications development and/or application to clinical practice. However, with a large unmet medical need and an estimated market size of several hundred million dollars per year, if developed, biomarkers for cocaine dependence will hold tremendous value to both industry and public health.

Modeling Substance Abuse for Applications in Proteomics

The ability to model aspects of human addictive behaviors in laboratory animals provides an important avenue for gaining insight into the biochemical alterations associated with drug intake and the identification of targets for medication development to treat addictive disorders. The intravenous self-administration procedure provides the means to model the reinforcing effects of abused drugs and to correlate biochemical alterations with drug reinforcement. In this chapter, we provide a detailed methodology for rodent intravenous self-administration and the isolation and preparation of proteins from dissected brain regions for Western blot analysis and high-throughput proteomic analysis. Examples of cocaine-induced alterations in the abundances of ionotropic glutamate receptor subunits in reinforcement-related brain regions are provided.

Sudden-Onset Behavioral Disturbance in a Woman with No Prior Psychiatric, Medical History

Mrs. M is a 30-year-old, married, African-American woman with no prior history of mental illness who was initially admitted to a general hospital due to changes in behavior. Mrs. M’s husband reported that during the 2 days prior to her admission, she seemed to have difficulty paying attention and registering information; she would sometimes talk excessively and at other times would stare blankly and not engage in any conversation with her husband. She also experienced insomnia and was intermittently restless. When she initially arrived at the general hospital, she was agitated and was given a one-time dose of ziprasidone 20 mg orally to calm her down. While at the general hospital, the patient was also selectively mute. The initial medical work-up in

Novel Methodologies: Proteomic Approaches in Substance Abuse Research

Proteomics is the study of sequence, expression, function, interaction and localization of proteins in tissues or fluids of various organisms. With the advent of proteomic techniques providing greater selectivity, sensitivity and throughput, drug abuse researchers are faced with decisions as to the selection of the most appropriate applications for their respective questions. In addition, drug abuse researchers face additional challenges that are unique to neuroscience limited sample quantities, heterogeneous cellular compositions of samples and the hydrophobic nature of large classes of proteins of interest. The purpose of this chapter is provide the drug abuse researcher with current technologies and methodologies for examining coordinated regulation of multiple proteins in tissue, primarily brain. Chapter content includes useful information on subcellular organelle isolation, protein fractionation and separation using two-dimensional gel electrophoresis and multi-dimensional liquid chromatography, methods for quantification of differential protein expression (e.g., two-dimensional difference in gel electrophoresis and isotope-coded affinity tags (ICAT and iTRAQ)), and mass spectrometry approaches. An overview of the techniques used currently to assign post-translational modification status on a proteomics scale is also evaluated. The application of these approaches to the study of cocaine and alcohol abuse is reviewed.