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Proteomics Module |
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Recent advance in technology is enabling the simultaneous global analysis of a large number of different genes, transcripts and proteins. This unprecedented analysis of changes in a large group of biological molecules is likely to provide unique insights into the pathways and interaction of these molecules and their role in the organization of cellular function.
With the recent development of expressed sequence tag (EST) database's and the sequencing of the human genome there has emerged methods for analyzing genome-wide expression using techniques such as DNA microarrays and oligonucleotide chips. As discussed in the Microarray description, once can use such methods for studying gene expression in the eye. The analysis of protein expression profiles provide additional information that can complement and overlap with these gene expression studies. For example, proteins can undergo dynamic (e.g. phosphorylation) and static modifications (e.g. disulphide bonds formation) that may not be apparent from transcript analysis. Consequently, the dual application of transcript and protein profiling will provide a more complete understanding of the regulation and interaction of the gene at multiple levels.
Proteomic research is a consecutive two-step procedure that presently relies on techniques for protein separation (such as 2D gels, subcellular fractionation) followed by analysis (e.g. mass spectrometry). Two-dimensional gel electrophoresis (2-D PAGE) is currently the best method for simultaneously complex mixtures of proteins that are commonly found in biological samples. The introduction of immobilized pH gradients (IPGs) has allowed 2-D PAGE analysis that is reproducible and due to an increase in sample loading, detection of lower abundance species. In this module, various investigators will use subcellular fractionation and/or 2D-PAGE to analyze mixtures of proteins by mass spectrometry. While silver stains have been commonly used to detect proteins on gels, it is known that they alkylate a and E-amino groups of protein. Modifications to the basic protocol have been utilized by various investigators to avoid this problem when further analysis has been required. Such modifications (e.g. elimination of glutaraldehyde) from the staining protocol however in general decrease the sensitivity of the stain and increase gel background. As an alternative, SYPRO Ruby Protein by 2D-PAGE and shown to be as sensitive as silver stain methods. Furthermore, SPYRO Ruby stain is fully compatible with MS thus facilitating protein identification after electrophoresis. At the Institute there is a protein and nucleic acid core facility that has a Pharmacia IPGphor IEF system for the first dimension and both Novex and Bio-Rad electrophoresis units for the second dimension. These services are provided on a fee-for-service basis and it is anticipated that a number of investigators will use this service. However, this facility lacks instrumentation for analysis of the 2D gels. To aid in the analysis of the 2D gels it is proposed to purchase a fluorescence imaging system (e.g. Amersham Pharmacia Typhoon 8600 or Fluorimager) and appropriate software for 2D gel analysis (e.g. Bio Rad Melanie III).
Completion of the human genome sequence has afforded many new technological opportunities for the study of the eye. Tremendous enthusiasm has been generated for the use of DNA microarrays, a resource created by the existence of sequences for human genes. As the human genome project has enabled the global study of gene transcription, this same sequence resource has enabled facile identification of proteins. Protein identification is possible through the generation of mass spectrometry data for digested proteins. The molecular weights of peptides can be accurately measured and this information, or pattern, can be compared to the predicted patterns produced by all the proteins in the database. If a preponderance of the peptide molecular weights match to a specific protein, then there is a high probability the proteins are the same. A second and more powerful approach relies on the fragmentation patterns produced by tandem mass spectrometry analysis of peptides. A distinct advantage to this approach is the ability to identify the components of mixtures and to specifically locate sites of covalent modification. Mass spectrometry approaches allow facile identification of proteins obtained from 1- or 2-D gel electrophoresis obtained from protein-protein interaction studies or proteins expressed in response to some disease state or stimulus. With the completion of a rough draft of the human genome, the analysis of proteins in human and mouse should be simplified.
This module will provide proteomic analysis capability to Core grant investigators. Researchers will be able to use to 2-DGE for the separation of complex mixtures of proteins, measurements that will complement studies preformed on DNA microarrays. Furthermore, researchers will be able to access state of the art mass spectrometry analyses to identify proteins.
Existing mass spectrometry
resources.
The Yates laboratory has expertise in the mass spectrometry analysis of
proteins from 1- and 2-Dimensional electrophoretic gels. We also have
extensive expertise with protein identification software and methods.
Proteins can be identified from silver stained gels and from unfractionated
mixtures. Existing resources available to the core of eye researchers
include a Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF),
Electrospray Ionization Time-of-Flight (ESI-TOF) and a microHPLC tandem
mass spectrometer (LC/MS/MS). We will provide mass spectrometry and protein
identification expertise to facilitate the identification of proteins
and modifications in vision related proteins. Dr. Yates laboratory will
provide computational resources and expertise for conducting the identifications.
For information regarding this site, contact Suzanne Bacon: bacons@scripps.edu
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