Mass spectrometry is comprised of a broad suite of advanced technologies important for identification of unknowns for most classes of biomolecules (and a large number of other molecules as well. Proteomics is broadly concerned with the global protein composition of cells, sub-cellular organelles, complexes and machines and how their class functions are modulated by posttranslational dynamics, e.g. kinome homeostasis and dynamics and the effects of molecular defects on aberrant signaling pathways and networks, etc.
Major efforts are being devoted to the characterization of complex mixtures of proteins using chromatography coupled mass spectrometry experiments, to detection and assignment of protein covalent modifications, to the analyses of modification-site-specific protein network kinetics/dynamics and to studies of intact protein complexes, e.g. pol II, nucleosomes, etc.
Mini Course Description:This course will focus on the practical aspects, i.e. experimental and spectral interpretation, involved in the identification of proteins and their posttranslational modifications. It will cover the fundamental principles of currently important mass spectrometry instrument platforms. It will provide an overview of key problems that are being tackled and solved at the protein level relevant to cell function/dysfunction; the detection and assignment of protein posttranslational modifications; and studies of site-specific dynamics/relative quantitation.
Methods for sample preparation and mass spectral data acquisition will be discussed together with how our UCSF bioinformatics toolbox (ProteinProspector) may be employed to facilitate the interpretation and presentation of mass spectral information.
Simultaneously, dry laboratory work is required to learn how to carry out the interpretation of complex mixtures of peptides isolated from cells involved in current active research projects. Initially blocks of time will be scheduled for class discussion of the experimental results you obtain.
|1 (Mon.4/3)||RJC||Protein Identification. Basics of peptide fragmentation processes. Database searching. How to measure the reliability of assignments.|
|2 (Wed.4/5)||SG||Mass Spectrometry Fundamentals: Ionization, Instrumentation; ion optics, resolution and mass accuracy; why these are important at protein vs peptide level.|
|3 (Mon.4/10)||JO||Sample preparation: Gels and Chromatography; IP/Affinity Tags, Digestion. What shouldn't be in the sample - Contaminants.|
|4 (Wed.4/12)||ALB||PTMs: Posttranslational modifications: Protein vs peptide analysis. PTM enrichment, PTM cross-talk.|
|5 (Mon.4/17)||AU||Quantitation strategies (SILAC, iTRAQ, MRM).|
|6 (Wed.4/19)||MT||Architecture of protein complexes and machines.|