Introduction to Proteomics 1 - University of Minnesota · Introduction to Proteomics 1.0 CMSP Workshop Tim Griffin Professor, BMBB ... •mass spectrometry •protein chips •yeast

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

Introduction to Proteomics 1.0

CMSP Workshop

Tim GriffinProfessor, BMBB

Faculty Director, CMSP


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


Alien language made understandable

CMSP Participants


Terminology made sensible

CMSP

Participants

NanoLC

MS/MSiTRAQ

monoisotopicESI

MALDI

b-ion

Precursor ionquadrupole

Stage-tip Ion trap

HCD TOF???Right on!


Who we areCenter 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


Who we are

Research AssociateInformatics Analyst Candace Guerrero

Postdoctoral 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

Julie KiriharaManager


‘Omic technologies and the molecular biology paradigm


Why proteomics and direct protein analysis?(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

Proteomics: A definition


• 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

Proteomics as a complement to genomics


• 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

Proteomic technologies and approaches


Enabling MS-based proteomics: “soft” ionization

Electrospray ionization(ESI)

Matrix-assisted laser desorption/ionization(MALDI)

• Making large, non-volatile biomolecules fly


ionization+

-+

+

+ -

--detectionseparation by m/z*

+ + quadrupole ion trap time-of-flight

MALDI Electrospray:

liquid chromatographynanospray

mass analysis of proteins, peptides

Nuts and bolts of mass spectrometry

*m/z = mass-to-charge


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


Image from ASMS 2014 workshop (Speaker: Haas)

Example of technology progress: more sensitive MS


The information currency of MS

200 400 600 800 1000 1200

m/z

Rel

ativ

e Ab

unda

nce


The “guts” of a mass spectrometer

ionization

m/z separation

m/z separation and detection

m/z separation and detection


Doing protein and proteomic analysis via MS

Sample preparation

Sample preparation MS analysisMS analysis Data analysisData analysis

Biological inquiryHypothesis

Experimental design

• 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


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


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!


base

pea

k in

tens

ity

time

organic concentration in mobile phase

The workhorse: LC-MS

• Separating molecular mixtures prior to introduction into MS


Some example applications: from simple to complex

Gygi, et. al. 1999, Molecular and Cellular Biology 19:1720

The “simple”: identifying a gel separated protein

2D gel electrophoresis: the original proteomics technology…but how to ID proteins?


Even the simple still requires care…..

In-gel digestion

Peptide purification

LC-MS/MS

Sequence Database Searching

• Process of identifying a gel-separated protein

Gygi, et. al. 1999, Molecular and Cellular Biology 19:1720


A bit more complicated: Identifying PTMs on a protein

• Phosphorylation• Glycosylation• Oxidations• Acetylation• Methylation• Lipid anchors• Ubiquitinylation/sumoylation

BUT….PTM analysis is not necessarily routine or easy!!

(abundance, enrichment, ionization, fragmentation….)

……

etc


Still more complicated: identification of proteins in complex mixtures• More complicated sample preparation (fractionation)


A bit more complicated: Quantitative proteomics

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

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


Quantitative proteomics: many methods available• Labeled versus un-labeled


Systems biology: integrating ‘omics data


Data acquisition

Raw data processing(Database searching)

Analysis of processed data(Statistical filtering, quantitative analysis)

Data organization and interpretation

Archiving and databasing

Dealing with the data: the rate-limiting step?


Workflow for protein identification


KEGG pathways

Bioinformatic interpretation and hypothesis generation


Thank you

Good luck

May all your ions fly well!

Documents

Introduction to Proteomics 1 - University of Minnesota · Introduction to Proteomics 1.0 CMSP Workshop Tim Griffin Professor, BMBB ... •mass spectrometry •protein chips •yeast