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Proteomics as a functional genomics tool.

To understand the function of all the genes in an organism, one needs to know not only which genes are expressed, when, and where, but also what the protein end products are and under which conditions they accumulate in certain tissues. Proteomics aims at describing the whole protein output of the genome and complements transcriptomic and metabolomic studies. Proteomics depends on extracting, separating, visualizing, identifying, and quantifying the proteins and their interactions present in an organism or tissue at any one time. All of these stages have limitations. Therefore, it is, at present, impossible to describe the whole proteome of any organism. Plants might synthesize many thousands of proteins at one time, and the whole potentially synthesized proteome certainly exceeds the number of estimated genes for that genome. This occurs because the gene products of one gene can differ due to alternative splicing and a variety of possible posttranslational modifications. It is, therefore, essential to optimize every step towards detecting the whole proteome while realizing the limitations. We concentrate here on the most commonly used steps in high-throughput plant proteomics with the techniques we have found most reproducible and with the highest resolution and quality.

Coloring Agents↗

Rice proteomics: a step toward functional analysis of the rice genome.

The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms and/or expression that occurs under stresses. In this study, the catalogues of the rice proteome were constructed, and a functional characterization of some of these proteins was examined. Proteins extracted from tissues of rice and proteins extracted from rice under various kinds of stress were separated by two-dimensional PAGE. An image analyzer was used to reveal a total of 10,589 protein spots on 10 kinds of two-dimensional PAGE gels stained by Coomassie Brilliant Blue. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The internal amino acid sequences of 633 proteins were determined using a protein sequencer or mass spectrometry after enzyme digestion of the proteins. Finally, a data file of rice proteins that included information on amino acid sequences and sequence homologies was constructed. The major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that turned out to be calreticulin and a gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have functions in the signal transduction pathway. The information thus obtained from the rice proteome will be helpful in predicting the function of the unknown proteins and will aid in their molecular cloning.

Agrobacterium tumefaciens↗

Proteomic identification and functional characterization of a novel ARF6 GTPase-activating protein, ACAP4.

ARF6 GTPase is a conserved regulator of membrane trafficking and actin-based cytoskeleton dynamics at the leading edge of migrating cells. A key determinant of ARF6 function is the lifetime of the GTP-bound active state, which is orchestrated by GTPase-activating protein (GAP) and GTP-GDP exchanging factor. However, very little is known about the molecular mechanisms underlying ARF6-mediated cell migration. To systematically analyze proteins that regulate ARF6 activity during cell migration, we performed a proteomic analysis of proteins selectively bound to active ARF6 using mass spectrometry and identified a novel ARF6-specific GAP, ACAP4. ACAP4 encodes 903 amino acids and contains two coiled coils, one pleckstrin homology domain, one GAP motif, and two ankyrin repeats. Our biochemical characterization demonstrated that ACAP4 has a phosphatidylinositol 4,5-bisphosphate-dependent GAP activity specific for ARF6. The co-localization of ACAP4 with ARF6 occurred in ruffling membranes formed upon AIF(4) and epidermal growth factor stimulation. ACAP4 overexpression limited the recruitment of ARF6 to the membrane ruffles in the absence of epidermal growth factor stimulation. Expression of GTP hydrolysis-resistant ARF6(Q67L) resulted in accumulations of ACAP4 and ARF6 in the cytoplasmic membrane, suggesting that GTP hydrolysis is required for the ARF6-dependent membrane remodeling. Significantly the depletion of ACAP4 by small interfering RNA or inhibition of ARF6 GTP hydrolysis by overexpressing GAP-deficient ACAP4 suppressed ARF6-dependent cell migration in wound healing, demonstrating the importance of ACAP4 in cell migration. Thus, our study sheds new light on the biological function of ARF6-mediated cell migration.

ADP-Ribosylation Factor 6↗

Comparative proteomics reveals distinct functions and localization of invasive Entamoeba histolytica and non-invasive Entamoeba moshkovskii proteins.

BACKGROUND: Entamoeba histolytica is a pathogenic protozoan accountable for amoebiasis, while Entamoeba moshkovskii is considered non-invasive. Despite morphological similarity, the molecular mechanisms underlying their different pathogenicity remain largely undefined. METHODS: Trophozoite proteins from axenic cultures of E. histolytica and E. moshkovskii were separated and identified using GeLC-MS/MS, and classified using Gene Ontology. Selected and differentially expressed proteins were validated by peptide-specific antibody production, ELISA, and immunofluorescence to determine cellular localization. RESULTS AND DISCUSSION: A total of 1,077 and 1,201 proteins were identified from E. histolytica and E. moshkovskii, respectively. The 801 of Entamoeba common proteins included kinases, GTPase-activating proteins, and heat shock proteins, reflecting conserved cellular processes. E. histolytica-unique proteins involved in nitrogen compound metabolism, vesicle-mediated transport, and catalytic activities, whereas E. moshkovskii proteins were related to lipid metabolism and environmental resilience. Subcellular localization revealed species-specific distribution of MmpL and AIG1-family proteins, suggesting potential roles in pathogenicity and host-immune response. A large proportion of hypothetical proteins was identified, highlighting gaps and opportunities for future study. CONCLUSIONS: Our study highlights conserved and divergent functions and cellular locations of Entamoeba species-specific proteins as insights for distinct pathogenicity and adaptation. MmpL and AIG1 proteins were proposed as potential targets for further diagnostic and therapeutic development.

Proteomics↗

Analyzing proteome topology and function by automated multidimensional fluorescence microscopy.

Temporal and spatial regulation of proteins contributes to function. We describe a multidimensional microscopic robot technology for high-throughput protein colocalization studies that runs cycles of fluorescence tagging, imaging and bleaching in situ. This technology combines three advances: a fluorescence technique capable of mapping hundreds of different proteins in one tissue section or cell sample; a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors; and a system for imaging the distribution of these protein clusters in a so-called toponome map. By analyzing many cell and tissue types, we show that this approach reveals rules of hierarchical protein network organization, in which the frequency distribution of different protein clusters obeys Zipf's law, and state-specific lead proteins appear to control protein network topology and function. The technology may facilitate the development of diagnostics and targeted therapies.

Dermatitis, Atopic↗

Expressing RNA aptamers inside cells to reveal proteome and ribonome function.

Small RNA molecules can participate in complex regulatory networks not merely through Watson-Crick interactions with other RNAs (as in anti-sense RNA), but also by forming discrete structures that bind protein or low-molecular weight targets. RNA aptamers derived from in vitro selections (SELEX) are being used inside cells for at least four purposes: (1) to antagonise normal cellular proteins as a means of elucidating their biological roles; (2) as decoys to natural RNA-binding proteins to reveal their functions or those of structural elements within naturally occurring transcripts; (3) as regulatory modules to govern the expression of exogenous genes; and (4) to antagonise disease-related targets for potential biomedical applications. This paper summarises recent advances in each of these areas, the parameters that influence successful application and the potential for future developments. The use of aptamers in vivo may serve as a paradigm for understanding how some non-coding RNAs and other elements of the ribonome exert their influence on the intracellular proteome, and is thus becoming an important tool for modern cell and molecular biology.

Animals↗

Proteome analysis and functional expression identify mortalin as an antiapoptotic gene induced by elevation of [Na+]i/[K+]i ratio in cultured vascular smooth muscle cells.

Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in remodeling of vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Recently, we have found that apoptosis in cultured VSMCs can be inhibited by inversion of the intracellular [Na+]/[K+] ratio after the sustained blockage of the Na+,K+-ATPase by ouabain. To understand the mechanism of ouabain action, we analyzed subsets of hydrophilic and hydrophobic VSMC proteins from control and ouabain-treated cells by 2-dimensional electrophoresis. Ouabain treatment led to overexpression of numerous soluble and hydrophobic cellular proteins. Among proteins that showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 (HSP70) superfamily and a marker for cellular mortal and immortal phenotypes. Northern and Western blotting and immunocytochemistry all have confirmed that treatment of VSMCs with ouabain results in potent induction of mortalin expression. Transient transfection of cells with mortalin cDNA led to at least a 6-hour delay in the development of apoptosis after serum deprivation. The expression of tumor suppressor gene, p53, in mortalin-transfected cells was delayed to the same extent, and the expressed protein showed abnormal perinuclear distribution, suggesting that p53 is retained and inactivated by mortalin. Our studies therefore define a new [Na+]i/[K+]i-responsive signaling pathway that may play an important role in the regulation of programmed cell death in VSMCs.

Animals↗

Laser capture microdissection: beyond functional genomics to proteomics.

Proteomics will drive biology and medicine beyond genomics, and can have a profound impact on molecular diagnostics. The posttranslational modifications of cellular proteins that govern physiology and become deranged in disease cannot be accurately portrayed by gene expression alone. Consequently, new technology is being developed to discover, and quantitatively monitor, proteomic changes that are associated with disease etiology and progression. In the past, proteomic technologies were restricted to tumor cell lines or homogenized bulk tissue specimens. This source material may not accurately reflect molecular events taking place in the specific cells of the tissue itself. This article describes a completely new class of proteomic-based approaches aimed at the identification and investigation of protein markers in the actual histologically defined cell populations that are immersed in heterogeneous diseased tissue. It is envisioned that these investigations will eventually lead to novel diagnostic, prognostic, or therapeutic markers that can be applied to monitor therapeutic toxicity or efficacy.

DNA, Complementary↗

Functional genomics and proteomics in the clinical neurosciences: data mining and bioinformatics.

The goal of this chapter is to introduce some of the available computational methods for expression analysis. Genomic and proteomic experimental techniques are briefly discussed to help the reader understand these methods and results better in context with the biological significance. Furthermore, a case study is presented that will illustrate the use of these analytical methods to extract significant biomarkers from high-throughput microarray data. Genomic and proteomic data analysis is essential for understanding the underlying factors that are involved in human disease. Currently, such experimental data are generally obtained by high-throughput microarray or mass spectrometry technologies among others. The sheer amount of raw data obtained using these methods warrants specialized computational methods for data analysis. Biomarker discovery for neurological diagnosis and prognosis is one such example. By extracting significant genomic and proteomic biomarkers in controlled experiments, we come closer to understanding how biological mechanisms contribute to neural degenerative diseases such as Alzheimers' and how drug treatments interact with the nervous system. In the biomarker discovery process, there are several computational methods that must be carefully considered to accurately analyze genomic or proteomic data. These methods include quality control, clustering, classification, feature ranking, and validation. Data quality control and normalization methods reduce technical variability and ensure that discovered biomarkers are statistically significant. Preprocessing steps must be carefully selected since they may adversely affect the results of the following expression analysis steps, which generally fall into two categories: unsupervised and supervised. Unsupervised or clustering methods can be used to group similar genomic or proteomic profiles and therefore can elucidate relationships within sample groups. These methods can also assign biomarkers to sub-groups based on their expression profiles across patient samples. Although clustering is useful for exploratory analysis, it is limited due to its inability to incorporate expert knowledge. On the other hand, classification and feature ranking are supervised, knowledge-based machine learning methods that estimate the distribution of biological expression data and, in doing so, can extract important information about these experiments. Classification is closely coupled with feature ranking, which is essentially a data reduction method that uses classification error estimation or other statistical tests to score features. Biomarkers can subsequently be extracted by eliminating insignificantly ranked features. These analytical methods may be equally applied to genetic and proteomic data. However, because of both biological differences between the data sources and technical differences between the experimental methods used to obtain these data, it is important to have a firm understanding of the data sources and experimental methods. At the same time, regardless of the data quality, it is inevitable that some discovered biomarkers are false positives. Thus, it is important to validate discovered biomarkers. The validation process may be slow; yet, the overall biomarker discovery process is significantly accelerated due to initial feature ranking and data reduction steps. Information obtained from the validation process may also be used to refine data analysis procedures for future iteration. Biomarker validation may be performed in a number of ways - bench-side in traditional labs, web-based electronic resources such as gene ontology and literature databases, and clinical trials.

Animals↗

Functional genomics and proteomics in control of breathing.

New genomic and proteomic techniques offer remarkable promise as tools to address longstanding questions regarding molecular mechanisms involved in the control of breathing. Here, these techniques are described. Additionally, recent examples of the application of these and related molecular techniques are provided-particularly including observations regarding the role of S-nitrosylation signaling reactions in regulating ventilatory responses.

Animals↗

Miniaturization in functional genomics and proteomics.

Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future.

Biological Assay↗

Functional and quantitative proteomics using SILAC.

Researchers in many biological areas now routinely characterize proteins by mass spectrometry. Among the many formats for quantitative proteomics, stable-isotope labelling by amino acids in cell culture (SILAC) has emerged as a simple and powerful one. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and - as a quantitative phosphoproteomics technology - directly uncovers important points in the signalling pathways that control cellular decisions.

Amino Acids↗

Gateway-compatible vectors for plant functional genomics and proteomics.

Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available "entry vector" and are then recombined into various "destination vectors" for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of "pEarleyGate" plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.

Amino Acid Sequence↗

Integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics.

Recent outbreaks of avian influenza in humans have stressed the need for an improved nonhuman primate model of influenza pathogenesis. In order to further develop a macaque model, we expanded our previous in vivo genomics experiments with influenza virus-infected macaques by focusing on the innate immune response at day 2 postinoculation and on gene expression in affected lung tissue with viral genetic material present. Finally, we sought to identify signature genes for early infection in whole blood. For these purposes, we infected six pigtailed macaques (Macaca nemestrina) with reconstructed influenza A/Texas/36/91 virus and three control animals with a sham inoculate. We sacrificed one control and two experimental animals at days 2, 4, and 7 postinfection. Lung tissue was harvested for pathology, gene expression profiling, and proteomics. Blood was collected for genomics every other day from each animal until the experimental endpoint. Gross and microscopic pathology, immunohistochemistry, viral gene expression by arrays, and/or quantitative real-time reverse transcription-PCR confirmed successful yet mild infections in all experimental animals. Genomic experiments were performed using macaque-specific oligonucleotide arrays, and high-throughput proteomics revealed the host response to infection at the mRNA and protein levels. Our data showed dramatic differences in gene expression within regions in influenza virus-induced lesions based on the presence or absence of viral mRNA. We also identified genes tightly coregulated in peripheral white blood cells and in lung tissue at day 2 postinoculation. This latter finding opens the possibility of using gene expression arrays on whole blood to detect infection after exposure but prior to onset of symptoms or shedding.

Animals↗