Proteomics

Proteomics is a rapidly advancing field of science that aims to characterise the proteins that are essential to all living things. Proteins are polymers of amino acids. Each type of protein is unique, identified by the sequence of amino acids that are assembled from coded genetic instructions. The sequence of amino acids then determine how the protein folds to form a complex three dimensional structure. This structure then determines the proteins’ function.

Proteins are the structural, regulatory and enzymatic workhorses of cells. Whichever biological process you investigate, proteins are responsible for the complex interplay between our genes and the environment. Proteins are responsible for converting sunlight into sugar, sugar into energy and energy into action. Proteins regulate our biological processes to ensure everything happens at the right time and in the right place. Even our genes are replicated, repaired and transmitted through the actions of proteins. While biology is the sum of many different chemicals and chemical processes, proteins stand out as the most diverse and functionally important class of molecules in biochemistry.

Genomics, Transcriptomics, Proteomics: why study proteins.

By sequencing genes and studying patterns of gene transcription, we can learn a great deal about biology and how genes influence biological outcomes. However, biology is sufficiently complex as to defy our attempts at understanding. While genes and gene transcripts are relatively easy to analyse, there is not a perfect correlation between genetics and the organisms that spring from those genes.

The reason behind this additional complexity is of course proteins. Our genes define our protein sequences but not the dynamic patterns of post-translational modification, intra-cellular trafficking or the rates of protein degradation that occur in response to our environment. Ultimately it is proteins that grant living things their flexibility. And it is only by studying the complex world of proteins that a greater understanding of biology can be achieved.

The power of proteomics

The global compilation of proteomic data has produced significant resources for biological researchers. The largest proteomic databases now include the sequences of over 5 million individual proteins. The interactome (the patterns of interactions between different proteins) of yeast and other species is being mapped at ever greater levels of resolution. Over 200,000 individual phosphorylation sites from almost 20,000 individual proteins have been identified. Increasingly, this information is collected into open-access databases that allow researchers the ability to search for answers to their particular biological questions. More importantly, this technology is now readily accessible to Victorian researchers interested in tackling the big questions regarding human disease, environmental management and agriculture.

Proteomics & Metabolomics Victoria is a coalition of researchers and industry partners interested in promoting proteomic and metabolomic techniques to Victorian scientists. Advances in instrumentation, particularly in the field of mass spectrometry, has made the field of proteomics increasingly powerful and productive. Where the techniques used to analyse proteins once required a high degree of training, pre-prepared kits and reagents have dramatically reduced developmental costs for research laboratories. For researchers interested in performing mass spectrometry experiments on instruments that can cost over a million dollars, the establishment of mass spectrometry core-facilities at Australian universities now provide economical access and expert advice for successful experimental design. Proteomics is available to enhance the research of all Victorian biological scientists.

Proteomic Analysis and Your Research

Gel Spot Analysis

The simplest form of proteomic analysis is the identification of proteins from polyacrylamide gels. Gel electrophoresis is commonly used for analysing proteins, often in combination with western blotting. But how many times have you asked yourself – what is that mysterious protein band and is it important? Protein bands stained with coomassie blue or silver stains can be simply identified by routine mass spectrometry analysis. When applied to co-immunoprecipitation experiments, gel electrophoresis and mass spectrometry can identify the protein interactions that regulate important biological processes.

2-Dimensional Gel Electrophoresis

2-Dimensional gel electrophoresis provides even greater resolution of the complex protein populations present in cells and tissues. Instead of resolving proteins purely in terms of size, 2-dimensional gel electrophoresis first separates proteins based on their isoelectric points, providing greater numbers of protein identifications and also identifying post-translational modifications such as phosphorylation. By simultaneously analysing two different protein populations labelled with different fluorescent probes, differences in protein expression and modification can be identified, a process known as Differences Gel Electrophoresis (DIGE). Again, individual proteins can be identified by routine mass spectrometry analysis.

Quantitative Proteomics with Mass Spectrometry

As mass spectrometers have become faster, modern instruments can now sequence up to 30 different peptides every second. Combined with efficient chromatography, complex protein mixtures can be digested into specific fragments using proteases and thousands of individual peptides identified in a single analysis. This process is known as bottom-up protein proteomics. Combining high-powered peptide identification with quantitation, now allows changes in protein expression to be identified in greater detail than ever before. A variety of different quantitation strategies are available, some allowing the multiplexing of up 8 different samples in the one mass spectrometry analysis.

Identification of Post-translational Modifications

Protein function is often dictated by post-translation modifications that occur during the lifetime of the protein. These modifications can include phosphorylation, acetylation, ubiquitination, glycosylation and proteolytic cleavage, to name a few. The identification of these modifications is critical to our understanding of how proteins, and hence biological processes, are regulated. Mass spectrometry provides to tools to identify and characterise these modifications in proteins and even measure the patterns of modification in response to the cells’ environment. Many post-translational modifications can now be identified efficiently so as to identify hundreds in a single analysis.

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