Thesis
Multiplexed fragmentation and protein interaction reporter technology application to human cells
Washington State University
Master of Science (MS), Washington State University
2009
Handle:
https://hdl.handle.net/2376/101828
Abstract
Chemical cross-linking combined with mass spectrometry is a feasible approach to study the protein-protein interactions. However, system complexity and cross-linking product heterogeneity have precluded widespread chemical cross-linking use for large-scale identification of protein-protein interactions. Mass spectrometry identifiable cross-linkers protein interaction reporters (PIRs) were developed to overcome these problems. PIR couple with mass spectrometry was applied to study the protein-protein interactions of prokaryotic cells, such as Shewanella oneidensis MR-1. However, the biological features of eukaryotic cells are different with prokaryotic cells. In this report, PIR coupled with mass spectrometry was applied to human cancer cells, such as MCF7 and HeLa cells. 109 proteins in MCF7 and 127 proteins in HeLa were labeled with PIRs and identified. The bottom-up LC/MS method involves the protein denaturation, protease digestion and LC/MS/MS to identify proteins and peptides. The most common data acquisition method for LC/MS/MS, can allow the identification of relatively abundant proteins. In biological systems, functional proteins, such as enzymes and membrane proteins are of relatively low abundance, thus data-dependent acquisition is generally insufficient to identify many functional proteins. Multiplexed fragmentation was developed. Multiplexed fragmentation with PIR strategy is a potential approach to study protein-protein interactions. However, ambiguous identification of PIR labeled peptides is the major challenge. To overcome this problem, this report demonstrated the feasibility of LC/3-stage multiplexed fragmentation approach to study protein-protein interactions with PIRs.
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Details
- Title
- Multiplexed fragmentation and protein interaction reporter technology application to human cells
- Creators
- Hye In Nam
- Contributors
- James E. Bruce (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Chemistry, Department of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; Pullman, Wash. :
- Identifiers
- 99900525056201842
- Language
- English
- Resource Type
- Thesis