Novel Methodologies: Proteomic Approaches in Substance Abuse Research

Acknowledgment

The writing of this chapter was supported in part by funding of the following NIH grants: DA012498, DA003628, and DA06634 (SEH).

Introduction

The comprehensive sequencing of human and other important genomes has enhanced our understanding of the cellular organization and function in higher organisms. This has been largely accomplished by the innovations in large-scale analysis of messenger RNA (mRNA) expression (microarrays, serial-analysis of gene expression [SAGE], and differential display). Genomics-based approaches have led to unprecedented advances in our understanding of the biological basis of substance abuse; however, the next step in systems biology is the examination of coordinate expression of the entire complement of proteins, including modifications and protein-protein interactions—proteomics. The broad-scale analysis of proteins in health and disease is essential given that proteins are central components of cellular physiology carrying out the greater part of biological events in the cell, even though certain mRNAs can act as effector molecules. Furthermore, it is important to note that mRNA and protein analyses are not interchangeable, with each being governed by distinct spatial, temporal, and physiological processes that generally prevent correlation of mRNA and protein expression in neuronal systems.

Proteomics involves the evaluation of the entire complement of proteins in a biological system with respect to structure, expression level, protein-protein interactions, posttranslational modifications (PTMs)—often referred to as structural, functional, and expression proteomics, respectively. The majority of early efforts in proteomics have been directed toward a comparison of differential protein expression and identification in disease and control tissues. However, changes in protein abundance do not define protein function exclusively, as many vital functions are brought about by PTMs, interactions among proteins, and differential distribution in subcellular components. Multiple proteomic strategies are needed to capture the involvement of regulatory mechanisms that affect protein abundance and function, such as protein-protein interactions and subcellular distribution.

The advent of proteomics can be attributed in part to the rapid development of mass spectrometry (MS), bioinformatics, and the current accessibility of vast protein databases from various organisms. These rapid advancements have improved our understanding of the cellular structure and function within the brain and the roles of various proteins and protein interactions in health and disease. However, the central nervous system poses unique challenges to proteomic inquiries, including the temporal and spatial expression characteristics of neurons and glia, the cellular heterogeneity of brain regions, the connectivity and communication between neurons, and the dynamic structural and functional alterations in neurons and glia that occur as a function of the interaction between the organism and the environment, development, learning and memory, and disease. These challenges can be overcome to some extent by combining specific isolation and fractionation procedures with high-throughput protein separation and analysis strategies to yield a more global view of the proteome in different physiological states than has been available previously. For example, before the advent of high-throughput proteomics technologies, our knowledge of protein alterations and the durations of those alterations induced by substance abuse was limited to fewer than 100 proteins—primarily expression levels of proteins assessed either individually or a few proteins at a time. With the development of proteomic technologies and strategies, it is now possible to evaluate significant portions of the neuroproteome (thousands of proteins) from crude homogenates to discrete cellular domains. Proteomic analysis strategies allow the simultaneous assessment of thousands of proteins of known and unknown function, thereby enabling a more comprehensive view of the protein orchestration in addictive disorders. Broad-scale evaluations of protein expression are well suited to the study of drug abuse, particularly in light of the complexity of the brain compared with other tissues, the multigenic nature of drug addiction, the vast representation of expressed proteins in the brain, and our relatively limited knowledge of the molecular pathology of this illness.

The development of innovative strategies has been ongoing in neuroproteomics, in particular for the study of PTMs, mapping of proteins from multiprotein complexes, and mapping of organelle proteomes. An understanding of the proteins in neurons along with their expression levels, their PTMs, as well as the protein-protein interaction maps would revolutionize addiction biology and addiction medicine in that we would then be able to expand our knowledge of the biochemical alterations specifically associated with substance abuse. Such information would be used to identify new targets for medication development.

Technology and Methods for Expression Proteomics

Protein Fractionation

The biological samples subjected to proteomic analysis in neuroscience include tissue, distinct cell populations, and cerebrospinal fluid (CSF). Each type of sample is extremely complex, as the protein constituents vary in charge, molecular mass, hydrophobicity, PTM, as well as spatial and temporal expression. The number of coding genes for the CNS fluctuate between 25,000 and 30,000. This added complexity of the neuroproteome will be overwhelming if we hypothesize that each protein on average has 10 splice variants, cleavage products, and PTMs, yielding approximately 250,000 to 300,000 protein isoforms to assess. Currently, there are no proteomic methods that have the capacity to separate and identify the entire proteome. One approach is to reduce the complexity of the proteome by subcellular fractionation procedures, thereby allowing a more thorough assessment of cellular domains (e.g., synapse, membrane, nucleus, and cytoplasm) while enriching low-abundance proteins that may not be detectable at the level of whole cell protein analysis.

Protein stability and purity as well as prevention of protein degradation and modification are of critical importance throughout various stages of proteomic analysis. Rapid removal of brain tissue, dissection, and freezing are imperative for the maintenance of the proteome state in the sample. Protease and phosphatase inhibitors are used to help prevent degradation and dephosphorylation of proteins during protein preparation ; however, care should be taken that adducts and charge trains are not introduced by these inhibitors. Purification of proteins from other cellular substances is also necessary, for example, lipids, several proteins (e.g., albumin and immunoglobulin are particularly abundant in the brain), and nucleic acids should be eliminated from the protein sample. The most common methods of purification rely on selective precipitation including acetone and trichloroacetic acid, although a number of commercially available kits are available.

Cerebrospinal Fluid

CSF is secreted by the choroid plexus in the lateral ventricles and is found in the cerebral ventricles and in the subarachnoid space flowing down the spinal canal as well as upward over the brain convexities. CSF, which an important determinant of the extracellular fluid (ECF) surrounding neurons and glia in the CNS, removes harmful brain metabolites, provides a mechanical cushion, and serves as a conduit for peptide hormones secreted by the hypothalamus. CSF is in steady state with the ECF and thus is considered to contain biochemical constituents that reflect neural activity.

Although proteomic studies of neuronal tissue have multiple challenges including the use of postmortem tissue and invasive biopsies from antemortem tissues, CSF proteomics is amenable to serial analysis by minimally invasive lumbar puncture. A change in the expression of CSF constituents may provide important insights into various CNS diseases by improving our understanding of the molecular basis of disease as well as providing disease biomarkers. Given the low protein concentration (∼150–450 μg/mL) and the high salt concentration (>150 mmol/L) of CSF and the abundance of albumin (∼60% of the total CSF protein) and immunoglobulin, it is necessary to deplete these abundant proteins (e.g., affinity removal, solid phase extraction) and reduce the salt concentration (e.g., protein affinity columns, ultrafiltration, and dialysis) to improve protein recovery and allow better detection of low-abundance proteins. This limitation, the depletion of some of the proteins of interest, can be overcome by a separate analysis of the depleted abundant proteins to ensure the analysis of proteins interacting with the abundant proteins.

Cellular Domain

Several recent proteomics studies have employed fractionation methods that allow collection of multiple cellular components from one tissue source. This allows a greater amount of each fraction to be used initially, thereby enabling analysis of low-abundance proteins. As the fractions are generated from the same samples, the experimental variability is reduced with the additional advantage of an additive increase in the whole proteome analyzed. The crucial drawback has been the overlap of the proteins between fractions.

Cytoplasm

Because the current proteomic strategies rely heavily on two-dimensional (2D) gel electrophoresis, which has been optimized for the analysis of soluble protein fractions, it is not surprising that most initial phases of proteomic analysis have focused on profiling of the cytoplasm. Most of the key regulators of the signaling pathways are housed in the cytoplasm, where, beside regulating the expression of receptors, they also channel important cytodynamic information between the nucleus and the membrane proteins. Some of the recent studies profiling the cytoplasm have revealed interesting new paradigms in our understanding of neurobiology.

Nucleus

The nucleus has a high degree of organization, consisting of structurally and functionally distinct compartments: nucleolus, nuclear speckles, nuclear pore complex, and nuclear envelope. The nucleus is a highly organized organelle consisting of domains that are fundamental for preserving the homeostasis of the cellular milieu. The profiling of the nuclear proteome in neuroscience has been the slowest of all subcellular fractions. However, there have been some good studies documenting the need to do so. In addition to the soluble fraction of nucleus, there has been an interest in other compartments of nucleus—nuclear envelope, nuclear pore complex, and nucleolus—although no studies to date have been published using such methods in addiction biology research.

Mitochondria

The mitochondria is a complex structure involved in fundamental processes, such as the tricarboxylic acid (TCA) cycle, β-oxidation of fatty acids, the urea cycle, electron transport, oxidative phosphorylation, apoptosis, and heme synthesis. Neuroproteomic analyses of the mitochondria have focused on the abundance in different brain regions. Data sets from mitochondrial proteomes from different species and tissues have documented 400–700 mitochondrial-associated proteins, which will enable scientists to better understand the mitochondrial machinery in health and disease.

Membrane

Membrane and the membrane-associated proteins constitute nearly a third of the cellular proteins and represent targets of approximately two-thirds of pharmaceutical agents. These proteins are involved in various cellular processes including signal transduction, cell adhesion, exocytosis, and metabolism and ion transport. Because membrane proteins are amphipathic, their hydrophobic nature makes them difficult to study and necessitates different strategies for analysis as compared to cytosolic proteins, for example. Therefore, although great strides have been made toward the analysis of soluble cellular proteins, the analysis of membrane proteins reported in proteomic analyses has been underrepresented. The traditional proteomic approach of two-dimensional gel electrophoresis (2DGE) has many limitations for analyzing membrane proteins —including the insolubility of hydrophobic proteins in a nondetergent sample buffer and inadequate alkaline-protein resolution. To a large extent, these issues can be overcome using a variety of combinations of liquid chromatographic separation techniques.

Synaptosomes and Postsynaptic Density

Synapses can be fractionated into synaptosomes as well as distinct pre- and postsynaptic components. Synaptosomes constitute the entire presynaptic terminal (including mitochondria and synaptic vesicles) and portions of the postsynaptic terminal (including postsynaptic membrane and postsynaptic density [PSD]). The study of synaptic proteomes is an important starting point in neuroscience for understanding complex brain functions, critical for understanding neuroplasticity as well as the neuropathology associated with drugs of abuse.

Synaptosomes are subcellular membranous structures formed during mild disruption of brain tissue. The shearing forces cause the nerve endings to break off and subsequent resealing of the membranes form the synaptosomes. The synaptosomes have a complex structure equipped with components of signal transduction, metabolic pathways, and organelles as well as structural components required for vesicular transport. Synaptosomes can be isolated from brain homogenate by differential and density-gradient centrifugation.

The postsynaptic density (or PSD) is a disk-like structure with a thickness of ∼30–40 nm and width of ∼100–200 nm. The most important structures associated with it are the cytoskeletal proteins, regulatory enzymes, and neurotransmitter receptors and associated proteins. These constitute a very highly structured framework with a definite association of the receptors and ion channels with the signaling molecules and the cytoskeletal elements to play an imperative role in signal transduction as well as synaptic plasticity. There are several available fractionation methods for isolation of the PSD.

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