Introduction to Proteomics 1.0 CMSP Workshop Tim Griffin Professor, BMBB Faculty Director, CMSP © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Why are we here? Objectives For participants: • Learn basics of MS-based proteomics • Learn what’s necessary for success using MS-based proteomics • Designing experiments; sample preparation; data analysis For CMSP staff: • Prepare users so they are equipped to have success working with CMSP • Manage expectations – what can these technologies do and not do © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Alien language made understandable CMSP Participants © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Terminology made sensible Stage-tip Ion trap b-ion MS/MS iTRAQ TOF NanoLC HCD quadrupole Right??? on! Precursor ion monoisotopic MALDI ESI Participants CMSP © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Who we are Center for Mass Spectrometry and Proteomics • Operated through the Department of Biochemistry, Molecular Biology and Biophysics • Serving biological MS-related research needs across UofM campus and external institutions/private companies • Fee-for-service Internal Service Organization (ISO) • Supported by all Colleges at UofM using CMSP and Office of Vice President for Research (plus variety of granting sources) • Extensive collaboration with Minnesota Supercomputing Institute/OIT/UMII • Primary mission to support research efforts at the University of Minnesota, but also train others in the use of advanced technologies and research approaches © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Who we are Julie Kirihara Manager Informatics Analyst Candace Guerrero PostdoctoralResearch Associate researcher • 150+ collective years of experience in biological MS; hundreds of scientific publications • Diverse expertise – design, sample preparation, instrumentation, data analysis • Experience with MANY sample types and research studies • Fish….gophers….periodontal bacteria…snake venom © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 ‘Omic technologies and the molecular biology paradigm © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Why proteomics and direct protein analysis? (Genomic sequencing is cheaper, faster, more comprehensive…why proteomics?) • DNA/RNA characterization cannot predict post-transcriptional events © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Proteomics: A definition “Proteomics includes not only the identification and quantification of proteins, but also the determination of their localization, modifications, interactions, activities, and, ultimately, their function.” -Stan Fields in Science, 2001. Alternatively: proteomics = high-throughput biochemistry © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Proteomics as a complement to genomics • measurement of protein response, which is not always indicated by mRNA response • post-translational modifications • macromolecular interactions • sub-cellular location • high-resolution structural and molecular characterization • integration with genomic/transcriptomic data to comprehensively characterize biological systems © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Proteomic technologies and approaches • two-dimensional gel electrophoresis • mass spectrometry • protein chips • yeast 2-hybrid • phage display • antibody engineering • high-throughput protein expression • high-throughput X-ray crystallography • cell imaging © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Enabling MS-based proteomics: “soft” ionization • Making large, non-volatile biomolecules fly Electrospray ionization Matrix-assisted laser desorption/ionization (ESI) (MALDI) © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Nuts and bolts of mass spectrometry + + - - ionization separation by m/z* detection - + + - + + MALDI quadrupole mass analysis of Electrospray: ion trap proteins, peptides liquid chromatography time-of-flight nanospray *m/z = mass-to-charge © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Many instruments, same underlying process Image from : http://www.medwow.com Image from: https://www.sdstate.edu/chem/mass-spec Image from: http://planetorbitrap.com Image from: http://planetorbitrap.com © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Example of technology progress: more sensitive MS Image from ASMS 2014 workshop (Speaker: Haas) © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 The information currency of MS Relative Abundance 200 400 600 800 1000 1200 m/z © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 The “guts” of a mass spectrometer m/z separation and detection m/z separation m/z separation and detection ionization © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Doing protein and proteomic analysis via MS Biological inquiry Hypothesis Sample Experimental design MS analysis Data analysis preparation • Workshop structured to follow this ordering • All aspects are important: each must be done well for success • Challenge: • technologies within each component always changing • interdisciplinary © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 The importance of sample preparation • Garbage in, garbage out •Protein mixtures isolated from biological sources are complex (hundreds to thousands of components) • Mass spectrometers have limited peak capacities requiring separation and fractionation of protein and peptide mixtures prior to analysis • Separation methods include: • gels • liquid chromatography • affinity chromatography • immunochromatography • selective enrichment by covalent chemistry © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Protein chemistry: a challenge • Proteins offer unique challenges compared to other biomolecules (e.g. nucleic acids): – Solubility – Abundance (no PCR!) – Chemical heterogeneity Each protein is a unique character! © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 The workhorse: LC-MS • Separating molecular mixtures prior to introduction into MS organic concentration in mobile phase base peak intensity time © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Some example applications: from simple to complex The “simple”: identifying a gel separated protein 2D gel electrophoresis: the original proteomics technology…but how to ID proteins? Gygi, et. al. 1999, Molecular and Cellular Biology 19:1720 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Even the simple still requires care….. • Process of identifying a gel-separated protein In-gel digestion Peptide purification Gygi, et. al. 1999, Molecular and Cellular Biology 19:1720 LC-MS/MS Sequence Database Searching © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 A bit more complicated: Identifying PTMs on a protein • Phosphorylation • Glycosylation • Oxidations • Acetylation • Methylation • Lipid anchors • Ubiquitinylation/sumoylation …… etc BUT….PTM analysis is not necessarily routine or easy!! (abundance, enrichment, ionization, fragmentation….) © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Still more complicated: identification of proteins in complex mixtures • More complicated sample preparation (fractionation) © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry