Chem 219: Protein Mass Spectrometry and Proteomics

April 22 - May 8, 2020

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 and elements as well). Proteomics is broadly concerned with gaining new knowledge on global protein composition of cells,sub-cellular organelles, complexes and machines, how their class functions are modulated by posttranslational dynamics, e.g. kinome or ubiquitylome/protease homeostasis, and the effects of molecular defects on aberrant signaling pathways and networks, etc.

Currently major efforts are being devoted to the discovery of new proteins in complex mixtures of proteins and this revealed by proximity biotinylation (bioID, APEX, BAR), 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 mass spectral interpretation, involved in the identification and quantitation of proteins and their covalent modifications. It will cover the fundamental principles of currently important mass spectrometry instrument platforms. It will provide an overview of key scientific 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. It will also cover studies on the architecture of protein complexes and machines.

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.

In addition to lectures, participants will be given datasets to analyze so they can learn how to carry out the interpretation of complex mixtures of peptides isolated from samples in current active research projects. Oral presentations of these results will be scheduled during the final week of the course.

Lecture Schedule: April 22 - May 8, 2020
Wednesdays and Fridays, 1:00 - 3:00pm
via ZOOM

Lecture Lecturer Topic
1 (Wed.4/22) RJC Protein Identification. Basics of peptide fragmentation processes. Database searching. How to measure the reliability of assignments.
2 (Fri.4/24) ALB Fundamentals: Ionization, Instrumentation; ion optics, resolution and mass accuracy; why these are important at protein vs peptide level.
3 (Wed.4/29) JO Sample preparation: Gels and Chromatography; IP/Affinity Tags, Digestion. What shouldn't be in the sample - Contaminants.
4 (Fri.5/1) AU Large scale quantification strategies (Label-free, SILAC, iTRAQ, PRM, neucode).
5 (Wed.5/6) JM Posttranslational modifications: Protein vs peptide analysis. PTM enrichment, PTM cross-talk.
6 (Fri.5/8) MT Architecture of protein complexes and machines (chemical crosslinking).

Lecturers

ALB - Al Burlingame
RJC - Robert Chalkley
JM - Jason Maynard
JO - Juan Oses
MT - Mike Trnka
AU - Anatoly Urisman

National Institute of General Medical Sciences Adelson Medical Research Foundation