Spelling suggestions: "subject:"fractionation"" "subject:"hypofractionation""
1 |
Global Proteomic Detection of Native, Stable, Soluble Human Protein ComplexesHavugimana, Pierre Claver 12 December 2012 (has links)
Protein complexes are critical to virtually every biological process performed by living organisms. The cellular “interactome”, or set of physical protein-protein interactions, is of particular interest, but no comprehensive study of human multi-protein complexes has yet been reported. In this Thesis, I describe the development of a novel high-throughput profiling method, which I term Fractionomic Profiling-Mass Spectrometry (or FP-MS), in which biochemical fractionation using non-denaturing high performance liquid chromatography (HPLC), as an alternative to affinity purification (e.g. TAP tagging) or immuno-precipitation, is coupled with tandem mass spectrometry-based protein identification for the global detection of stably-associated protein complexes in mammalian cells or tissues. Using a cell culture model system, I document proof-of-principle experiments confirming the suitability of this method for monitoring large numbers of soluble, stable protein complexes from either crude protein extracts or enriched sub-cellular compartments. Next, I document how, using orthogonal functional genomics information generated in collaboration with computational biology groups as filters, we applied FP-MS co-fractionation profiling to construct a high-quality map of 622 predicted unique soluble human protein complexes that could be biochemically enriched from HeLa and HEK293 nuclear and cytoplasmic extracts. Our network is enriched in assemblies consisting of human disease-linked proteins and contains hundreds of putative new components and novel complexes, many of which are broadly evolutionarily conserved. This study revealed unexpected biological associations, such as the GNL3, FTSJ3, and MKI67IP factors involved in 60S ribosome assembly. It is my expectation that this first systematic, experimentally-derived atlas of putative human protein complexes will constitute a starting point for more in depth, hypothesis-driven functional investigations of basic human molecular and cellular biology. I also note that my generic FP-MS screening approach can, and is currently, being applied by other members of the Emili laboratory to examine the global interactomes of other mammalian cell lines, tissues, sub-cellular compartments, and diverse model organisms, which should expand our understanding of proteome adaptations and association networks associated with cell physiology, animal development and molecular evolution.
|
2 |
Global Proteomic Detection of Native, Stable, Soluble Human Protein ComplexesHavugimana, Pierre Claver 12 December 2012 (has links)
Protein complexes are critical to virtually every biological process performed by living organisms. The cellular “interactome”, or set of physical protein-protein interactions, is of particular interest, but no comprehensive study of human multi-protein complexes has yet been reported. In this Thesis, I describe the development of a novel high-throughput profiling method, which I term Fractionomic Profiling-Mass Spectrometry (or FP-MS), in which biochemical fractionation using non-denaturing high performance liquid chromatography (HPLC), as an alternative to affinity purification (e.g. TAP tagging) or immuno-precipitation, is coupled with tandem mass spectrometry-based protein identification for the global detection of stably-associated protein complexes in mammalian cells or tissues. Using a cell culture model system, I document proof-of-principle experiments confirming the suitability of this method for monitoring large numbers of soluble, stable protein complexes from either crude protein extracts or enriched sub-cellular compartments. Next, I document how, using orthogonal functional genomics information generated in collaboration with computational biology groups as filters, we applied FP-MS co-fractionation profiling to construct a high-quality map of 622 predicted unique soluble human protein complexes that could be biochemically enriched from HeLa and HEK293 nuclear and cytoplasmic extracts. Our network is enriched in assemblies consisting of human disease-linked proteins and contains hundreds of putative new components and novel complexes, many of which are broadly evolutionarily conserved. This study revealed unexpected biological associations, such as the GNL3, FTSJ3, and MKI67IP factors involved in 60S ribosome assembly. It is my expectation that this first systematic, experimentally-derived atlas of putative human protein complexes will constitute a starting point for more in depth, hypothesis-driven functional investigations of basic human molecular and cellular biology. I also note that my generic FP-MS screening approach can, and is currently, being applied by other members of the Emili laboratory to examine the global interactomes of other mammalian cell lines, tissues, sub-cellular compartments, and diverse model organisms, which should expand our understanding of proteome adaptations and association networks associated with cell physiology, animal development and molecular evolution.
|
Page generated in 0.0839 seconds