In this webinar, Kathryn Lilley from University of Cambridge will describe hyperLOPIT; a spatial proteomics method which enables the characterization of protein subcellular localization. Many proteins exist in highly controlled micro-environments and thus vary in a context-dependent manner increasing their functional diversity. Our understanding of the proteins residing in these niches is important to the analysis of protein function and knowledge of cellular systems. Moreover, the ability to chart the dynamic change in location is vital to the elucidation of the full repertoire of mechanisms that exist at the cellular level.
Using a combination of biochemical fractionation of mouse pluripotent embryonic stem cells, TMT 10-plex labeling, SPS-MS3 method and semi-supervised machine-learning tools, the hyperLOPIT workflow has resulted in the simultaneous assignment of the steady state location of over 6000 proteins to multiple subcellular compartments in a single experiment. The resulting map gives intriguing insight into stem cell biology, including information about the steady location of components of six major signaling pathways and the unexpected distribution of proteins involved in trafficking and the secretory pathway.
Key learning objectives
- To explain the hyperLOPIT method and its application to create a high resolution subcellular map of more than 6000 thousand proteins in a mouse embryonic stem cell line.
- To demonstrate the significant impact of SPS-MS3 workflow on the quantitative accuracy of 10plex TMT in conjunction with a Thermo ScientificTM Orbitrap FusionTM TribridTM Mass Spectrometer.
- To reinforce the importance of employing quantitative pipelines which apply robust data analysis.
Who should attend
- Mass spectrometrists involved in multiplexed quantitative proteomics studies.
- Proteomics facilities with projects aimed at deriving subcellular localization of proteins.
- Cell biologists with an interest in subcellular maps of protein localization.
Kathryn received her PhD in Biochemistry from the University of Sheffield in 1990. She then continued her scientific career as the manager of the Protein and Nucleic Acid Laboratory core facility at the University of Leicester for 11 years, initially starting with Edman degradation, DNA synthesis and HPLC core services and adding peptide synthesis, DNA sequencing and both MALDI-TOF and LC-MS mass spectrometry facilities during this period. In November 2000, she established the Cambridge Center for Proteomics (CCP) of which she is director which sits within the Cambridge Systems Biology Centre, University of Cambridge. This state-of-the-art center collaborates with a large number of groups in the UK and worldwide. The research focus of her group includes the advancement and application of methods in quantitative proteomics, especially using these techniques to accurately determine the location of proteins within sub-cellular structures and their interacting partners in studies involving cell differentiation and development.
Her research also involves the creation of novel informatics solutions for interrogating complex proteomics datasets and she thus collaborates with a number of computer scientists and biostatisticians. She also interacts with transcriptomics, and metabolomics facilities on large-scale system biology approaches. She was appointed to a personal Professorship in Cellular Dynamics in the Department of Biochemistry, University of Cambridge in 2012.