Proteomics in alcohol research

Abstract

The proteome is the complete set of proteins in an organism. It is considerably larger and more complex than the genome--the collection of genes that encodes these proteins. Proteomics deals with the qualitative and quantitative study of the proteome under physiological and pathological conditions (e.g., after exposure to alcohol, which causes major changes in numerous proteins of different cell types). To map large proteomes such as the human proteome, proteins from discrete tissues, cells, cell components, or biological fluids are first separated by high-resolution two-dimensional electrophoresis and multidimensional liquid chromatography. Then, individual proteins are identified by mass spectrometry. The huge amount of data acquired using these techniques is analyzed and assembled by fast computers and bioinformatics tools. Using these methods, as well as other technological advances, alcohol researchers can gain a better understanding of how alcohol globally influences protein structure and function, protein-protein interactions, and protein networks. This knowledge ultimately will assist in the early diagnosis and prognosis of alcoholism and the discovery of new drug targets and medications for treatment.

Figure 1

Flowchart showing the process of protein identification through a combination of two-dimensional gel electrophoresis (2–DE) with mass spectroscopy (MS). If the matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI–TOF MS) approach does not result in protein identification, additional analyses, such as electrospray ionization (ESI) combined with at least two steps of MS, may be used.

Figure 2

Example of a separation of human liver proteins by two-dimensional gel electrophoresis. Proteins were separated according to their isoelectric point (pI 4.0–6.5, acidic proteins) on the X axis, and their molecular weight (M_r 10–200 kDa) on the Y axis. Known proteins are labeled, but each spot might represent more than one unresolved protein. Multiple spots adjacent to each other have the same label because they represent post-translational modifications of the same protein, having the same molecular weights but different isoelectric points. SOURCE: Taken from SWISS-2DPAGE maps at http://us.expasy.org/ch2dothergifs/publi/liver-acidic.gif

Figure 3

(A) Example of a matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) peptide mass spectrum. Identified trypsin-derived peptides are marked with a filled circle. This analysis identified a total of 25 tryptic peptides with a mass:charge ratio (m/z) between 800 and 4,000 Da. (B) A graphical representation of the result of a database search showing the protein identified. Peptides that matched the sequence of that protein appear in dark red, and sequences that were covered by overlapping peptides are shown in yellow. The 25 identified peptides cover 28 percent of the protein sequence. The data obtained suggest that the protein being studied is the protein identified in the database search. SOURCE: Reprinted from Mann et al. 2001, with permission of the authors.

Figure 4

Schematic representation of the principle underlying the two-hybrid system for detecting in vivo protein–protein interactions. The assay is based on the fact that the transcription of the reporter gene is regulated by the activity of a specific protein (i.e., a transcription factor). Transcription factors are modular proteins consisting of two domains, a DNA-binding domain B and an activating domain A (see inset). To test if a known protein X interacts with a series of proteins Y (e.g., Y₁, Y₂, etc.), fusion proteins are genetically engineered in which domain A is fused to X (hybrid II) and domain B is fused to the Y proteins (hybrid I). Neither domain in the hybrid molecules thus generated can activate transcription alone if proteins X and Y do not interact (see upper panel). Only if proteins X and Y interact can domains A and B come close together so that the reporter gene can be transcribed (see lower panel). A similar approach can also be used to screen for complex interactions of three proteins (i.e., three-hybrid system) or for interactions between a protein and nucleic acids (i.e., one-hybrid system).

Figure 5

Example of a proteome microarray carrying 5,800 unique yeast proteins, which represents the entire yeast proteome. The enlarged area shows one of the 48 blocks containing 288 protein dots each. A minimum of 10 femtograms of protein is deposited per dot and detected as bright color (the lighter dots). The yeast proteome in the microarray is further tested for protein–protein interactions with known proteins of interest that carry another fluorescent color. NOTE: One femtogram is 10⁻¹⁵ g. SOURCE: Redrawn from Zhu et al. 2001, with permission of the authors.