Introduction to Proteomics 1.0

CMSP Workshop

Tim Griffin Professor, BMBB Faculty Director, CMSP

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

CMSP Participants

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

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

(Genomic sequencing is cheaper, faster, more comprehensive…why proteomics?)

• DNA/RNA characterization cannot predict post-transcriptional events

“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

• 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

• 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

• Making large, non-volatile biomolecules fly

Electrospray ionization Matrix-assisted laser desorption/ionization (ESI) (MALDI)

+ + - - 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

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

m/z separation and detection

m/z separation

m/z separation and detection

ionization

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

• 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!

• 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)

S. cerevisiae cell cycle (compliments of J.A. Huberman)

• Protein abundance is dynamic in response to environmental, genetic, biochemical, pathological perturbations.

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Quantitative proteomics: many methods available • Labeled versus un-labeled

Data acquisition

Raw data processing (Database searching)

Analysis of processed data (Statistical filtering, quantitative analysis)

Data organization and interpretation

Archiving and databasing

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Bioinformatic interpretation and hypothesis generation

KEGG pathways

Good luck

May all your ions fly well!