Abstracts


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Affinity Purification and Mass Spectrometry: An Attractive Choice to Investigate Protein-Protein Interactions in Plant Immunity
Ruiqiang Chen, Caren Chang, Mark L. Tucker and Bret Cooper

The use of affinity purification to isolate protein complexes from biological tissues, followed by mass spectrometry (AP-MS), has ballooned in recent years due to improvements in affinity purification protocols, sizeable increases in nucleic acid sequence data essential for interpreting mass spectra, and technological advances in mass spectrometry. Plant biologists are now exploiting AP-MS to identify plausible protein-protein interactions crucial to plant defense systems. As a result, knowledge of protein interactions in plants has grown. For example, new protein partners have been found to interact with RIN4 and RPS2, two plasma membrane-bound proteins critical for defense responses in Arabidopsis thaliana. Moreover, a nuclear protein complex in A. thaliana that includes the defense signaling protein MOS4 has been affinity purified and many of the identified protein partners found to be conserved with those in a protein complex previously characterized in yeast and humans. In another example, several proteins were identified that interact with a defense signaling GTPase, Rac1, in rice (Oryza sativa). Clearly, AP-MS is an important technique that will continue to provide novel insight into protein-protein interaction networks in plants. Here we review some of these recent discoveries and summarize the different techniques of AP-MS that have been used successfully to identify some of the interacting proteins in the plant defense response to pathogen attack.


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Application of Proteomic Techniques to Fruits and Vegetables
Jun Song and Gordon Braun

Proteomics and the technology supporting this science are proving to be invaluable in elucidating the tremendous complexity of biological processes. Among proteomic techniques, two-dimensional electrophoresis (2-DE) has been applied to resolve thousands of proteins and 2-DE has been especially useful for comparative studies between paired samples or populations. Improved protein extraction and purification protocols for recalcitrant fruits and vegetables have resulted in 2-DE techniques appearing in almost all fruit and vegetable proteomic studies currently being published. Numerous proteins involving various metabolic pathways have already been reported for tomato, pepper, strawberry, grape, banana, apple and pear. Significant improvements have been made to both gel and non-gel based proteomic research platforms. Better quantitative analyses and higher throughput proteomic technologies will further improve fruit and vegetable proteomic research and will have a significant impact on future breeding and post-harvest handling technologies to improve nutritional and eating quality.


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Characterization of Plant-Bacterial Interactions Using Proteomic Approaches
Zhenyu Cheng, Owen Z. Woody, Bernard R. Glick and Brendan J. McConkey

The explosion in the amount of genomic information available has revolutionized almost every aspect of the life sciences including proteomics. In tandem with advances in genomics, considerable developments in proteomic tools and technologies have greatly facilitated the application of proteomics to tackle numerous biological questions. Many measurable characteristics of proteins, including expression levels, cellular distributions, interactions with other molecules, and post-translational modifications, influence and direct their functions in various cellular processes. Proteomic techniques, such as comparative proteomics, mass spectrometry-based identification of post-translational modifications, and protein arrays, can be employed to study these aspects of proteins in a “host plus microbes” setting and in turn shed light on their interactions. This review summarizes the proteomic techniques applicable to host-microbial relations, with a particular focus on plant-bacterial interactions. This article also provides a comprehensive overview of the applications of proteomics in plant-bacterial interactions, including some of the most recent progress in the field.


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Computational Analysis of Amino Acid Mutation: A Proteome Wide Perspective
Jiajia Chen and Bairong Shen

Amino acid mutations may have diverse effects on protein structure and function. Thus reliable information about the protein sequence variations is essential to gain insights into disease genotype-phenotype correlations. With the recent availability of the complete genome sequence and the accumulation of variation data, determining the effects of amino acid substitution will be the next challenge in mutation research. The molecular consequences of amino acid mutations can readily be predicted by numerous bioinformatic methods, which analyze the mutation effects from different points of view. In this review, these approaches are categorized according to their analysis principles. The applicability of these tools for inference of mutation-structure-function relationship is also recapitulated. When the human diseases are likely to involve defects in multiple genes, most of the current mutation analysis focuses on single point mutation and lacks an expansive proteome-wide perspective. We propose in this review the application of the existing computational tools in the analysis of correlated mutations at a system level. Directions for future developments and implications are discussed, which will help to understand the networks underlying human disease.


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Detection of Protein-Protein Interactions Using Protein-Fragment Complementation Assays(PCA)
Emma Barnard, Neil V. McFerran, John Nelson and David J. Timson

Protein-protein interactions play a central role in many cellular processes. Their characterisation is necessary in order to analyse these processes and for the functional identification of unknown proteins. Existing detection methods such as the yeast two-hybrid (Y2H) and tandem affinity purification (TAP) method provide a means to answer rapidly questions regarding protein-protein interactions, but have limitations which restrict their use to certain interaction networks; furthermore they provide little information regarding interaction localisation at the subcellular level. The development of protein-fragment complementation assays (PCA) employing a fluorescent reporter such as a member of the green fluorescent protein (GFP) family has led to a new method of interaction detection termed Bimolecular Fluorescent Complementation (BiFC). These assays have become important tools for understanding protein interactions and the development of whole genome interaction maps. BiFC assays have the advantages of very low background signal coupled with rapid detection of protein-protein interactions in vivo while also providing information regarding interaction compartmentalisation. Modified forms of the assay such as the use of combinations of spectral variants of GFP have allowed simultaneous visualisation of multiple competing interactions in vivo. Advantages and disadvantages of the method are discussed in the context of other fluorescence-based interaction monitoring techniques.


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Determination of Binding Constant and Stoichiometry for Antibody-Antigen Interaction with Surface Plasmon
Shiming Lin, Adam Shih-Yuan Lee, Chih-Chen Lin and Chih-Kung Lee

A surface plasmon resonance (SPR) biosensor technology has recently been applied biochemically and clinically to the study of immunologic recognition and the evaluation of binding parameters for various interactions between antibodies (Abs) and antigens (Ags) at liquid-solid interface. The simple interaction between hapten and Ab fragment, e.g., variable single-chain fragment and antigenbinding fragment, can be described sufficiently by a 1:1 stoichiometry in SPR. However, the determination of the binding constant of an anti-protein Ab is usually complicated by the multivalence of the protein Ag. The SPR-based method enables direct determination of binding constants for a variety of specific Ab-Ag interactions in real-time. It also allows estimation of the binding stoichiometry and binding ratio for low-, intermediate-, and high-affinity Ab-Ag interaction systems. The present review is designed to indicate the theoretical background of SPR-based biosensor technology as well as to present the great variety of measurement modes of interaction kinetics that can be performed with these techniques. Quantitative aspects of the Ab-Ag interaction kinetics are reviewed, focusing especially on mono- and multi-valent Ab-Ag interaction modes using a SPR biosensor. Four model binding systems developed recently for use with SPR biosenser are described with principles and examples: (i) one to one interaction mode, (ii) nonequivalent two-site interaction mode, (iii) multiple equivalent-site interaction mode and (iv) multisite interaction mode. This article closes with two descriptions of the determinations of the binding stoichiometry and maximum binding ratio of Ab-Ag interactions.


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Functional Proteomics Using Direct Protein Inactivation
B. K. Eustace and D. G. Jay

There is a great need for high-throughput methods to help assign function to the proteins in the cell. In humans, there are ~30,000 genes that may be expressed in any particular cell type resulting in > 1 million distinct proteins. However, there are few functional proteomic methods to comprehensively study these proteins. One such method to address the cellular function of proteins is CALI (Chromophore-Assisted Laser Inactivation). CALI uses the specificity of antibodies (or other protein binding reagents) and the energy generated from excited dye molecules to specifically damage proteins. FALI (Fluorophore-Assisted Light Inactivation) is a modification of CALI that allows high-throughput protein disruption with a diffuse light source to inactivate samples simultaneously in multiwell plates. FALI is currently being used with large libraries of monoclonal or recombinant antibodies to identify proteins involved in tumor cell invasion, dispersal, and apoptosis. However, new innovations in light-mediated protein inactivation continue to be developed, and provide novel strategies for proteomics. GFP-CALI (Green Fluorescent Protein) and FlAsH-FALI (Fluorescein-Arsenical Helix) are two examples of such innovations. These methods use genetically encoded fusion proteins to direct lightinduced damage to the protein. So far these methods have been used in hypothesis-driven experiments, but they are readily adaptable to high-throughput proteomic screens. In this review, we will describe the current applications of CALI and FALI, and speculate on the future of direct light-induced protein inactivation for functional proteomics.


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Glycosite Analysis in Glycoproteomics by Mass Spectrometry
Yaohan Chen, Yang Zhang and Pengyuan Yang

Glycoproteomic researches have been rapidly progressing in the last few years. Although intact glycopeptide analysis which obtains glycosite and corresponding glycan information simultaneously is still challenging, various studies, combining glycosylation enrichment methods and mass spectrometry techniques, have accumulated a considerable amount of protein glycosite data for either N- or O-glycosylation or O-GlcNAcylation. This review will majorly focus on recent research outputs in the field of glycosite exploration.


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Mass Spectrometry in the Elucidation of the Glycoproteome of Bacterial Pathogens
Robert L.J. Graham and Sonja Hess

Presently some three hundred post-translational modifications are known to occur in bacteria in vivo. Many of these modifications play critical roles in the regulation of proteins and control key biological processes. One of the most predominant modifications, N- and O-glycosylations are now known to be present in bacteria (and archaea) although they were long believed to be limited to eukaryotes. In a number of human pathogens these glycans have been found attached to the surfaces of pilin, flagellin and other surface and secreted proteins where it has been demonstrated that they play a role in the virulence of these bacteria. Mass spectrometry characterization of these glycosylation events has been the enabling key technology for these findings. This review will look at the use of mass spectrometry as a key technology for the detection and mapping of these modifications within microorganisms, with particular reference to the human pathogens, Campylobacter jejuni and Mycobacterium tuberculosis. The overall aim of this review will be to give a basic understanding of the current ‘state-of-the-art’ of the key techniques, principles and technologies, including bioinformatics tools, involved in the analysis of the glycosylation modifications.


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Molecular Beacon Aptamers for Protein Monitoring in Real-Time and in Homogeneous Solutions
Zehui Cao, Steven W. Suljak and Weihong Tan

Aptamers are nucleic acids selected for binding target molecules of interest with high affinity and selectivity. They have seen increasing application in protein detection due to many of their advantages over traditional protein probes such as antibodies. Aptamers' robust yet flexible functional structures and relatively small sizes have allowed us to develop several strategies for sensitive protein detection in real time and in homogeneous solutions while posing minimum effects on the biological activities of the proteins. Quantitative protein analyses were done using aptamers labeled with a fluorophore and a quencher based on fluorescence resonance energy transfer (FRET), or using aptamers labeled with only one fluorophore based on fluorescence anisotropy. Real world biological samples were tested for the presence of target proteins. We believe that aptamers hold great potential for high throughput protein analysis in areas such as disease diagnosis and functional proteomics.