introduction to proteomics susan liddell university of nottingham [email protected] pgt...

Introduction to ProteomicsSusan Liddell

University of [email protected]

PGT short course 2014UoN Graduate School Course Post-genomics and bio-informatics

mailto:[email protected]

Sutton Bonington Proteomics labsDivision of Animal Sciences – South lab

Susan Liddell, Ken Davies

• Supports proteomics studies & collaborative projects– gel electrophoresis (mainly 2D)– protein identification via tandem MS

• Wide variety of types of projects and organisms – including some species with unreported genome sequences

human cow horse fungi bacteria archaea plants

Dr Ken Davis

Overview

• what is proteomics?• why study the proteome• proteomic strategies

– the 2D gel standard workflow– high throughput LC-MSMS

• draft human proteome• challenges

Biochem. J. (2012) 444, 169–181

Proteomics: an explosive growth in interest & expectations

Same search on

11/2012 36,547 entries

12/2013 44,947 entries

11/2014 51,150 entries

The NCBI database PubMed was searched using the term

“proteomics”

The increase in proteomics publications

Driving forces for proteomics

‒ Technical advances in 2D gel electrophoresis

IPG strips, multigel runners, buffer components

‒ Enormous advances in MS instrumentation

speed, sensitivity, resolution, soft ionisation

‒ Computer algorithms for searching databases with MS data in correlative based approaches to identify proteins

‒ Nucleotide databases with complete genome sequences

ProteomeThe term “proteome” was coined by a PhD student, Marc Wilkins

“the entire PROTEin complement expressed by a genOME of a cell or organism”

Wasinger et al 1995 Electrophoresis: 16:1090

Proteomics“...the identification of all the proteins encoded in the human genome....”

including modification, quantification, localisation and functional analysis for every cell type

Human Proteome Organisation (www.hupo.org)

http://www.hupo.org/

Proteomics

study of proteins and protein function usually on a genome wide scale

large scale or systematic characterization of the proteins of a cell, tissue, biofluid, or simple organism

Proteomics preceded genomics Human Protein Index N & L Anderson 1982

Aims of Proteomics

Global (unbiased) analysis of complex protein samples

Find changes in protein expression (potential biomarkers) in different biological situations (disease)

Development of diagnostic tools and therapeutic agents/drugs

Fundamental understanding of biological processes and mechanisms

proteins are the functional molecules in the cell and take part in virtually every cellular process

Why study proteins?

-catalysis-digestion, metabolism, DNA replication and repair, transcription, etc

-structure and movement-extracellular proteins are critical components of cartilage-muscle fibres are made up primarily of the proteins myosin & actin

-transporters-apolipoprotein A-1 carries lipids in the blood-haemoglobin carries oxygen

-proteins are the targets of the majority of drugs

-cell signalling, signal transduction, cell cycle, immunity

-the list goes on and on and on.....

Why analyse the proteome? Genome considerations

the genome provides only the blueprint – an inventory of the genes that could be expressed in a cell

• genomes are (largely) static

• proteomes vary enormously‒ cell types, developmental stage, environment, etc

• proteomes are highly dynamic, are constantly changing

• the proteome is a lot more complex than the genome, because most proteins are chemically modified and so have multiple forms

Images courtesy of en.wikipedia.org


Functional Annotation of the Arabidopsis Genome Using Controlled VocabulariesPlant Physiology (2004) Vol.135, p745

Arabidopsis genome annotationfunctional characterisation

26% molecular function unknown

sequence alone does not reveal biological function


• gene rearrangements‒ immunoglobulin heavy and light chains‒ T-cell receptor α and β chains

Why analyse the proteome? Transcript considerations

• RNA splicing

alternative splicing in a conserved family of ser/arg rich proteins in Arabidopsis generates up to 95 transcripts from only 15 genes


poor correlation between mRNA levels and

protein expression levels?– Comprehensive mass-spectrometry-based proteome quantification of

haploid versus diploid yeast. De Godoy et al 2008 Nature 455:1251

– “Overall correlation of mRNA and protein changes was poor....”

– “the relationship between mRNA and protein levels depends on the proteins investigated”

– Correlation between protein and mRNA abundance in yeastGygi et al 1999 MolCellBiol 19:1720

– A comparison of selected mRNA and protein abundances in human liverAnderson and Seilhamer 1997 Electrophoresis 18:533

Common covalent modifications of proteins affecting activity

Modification Donor moleculeExample of modified protein

Protein function

Phosphorylation ATPGlycogen phosphorylase

Glucose homeostasis; energy transduction

Acetylation Acetyl CoA HistonesDNA packing; transcription control

Myristoylation Myristoyl CoA Src Signal transduction

ADP-ribosylation NAD RNA polymerase Transcription

-Carboxylation HCO3- Thrombin Blood clotting

Ubiquitination Ubiquitin Cyclin Control of cell cycle

Biochemistry. Jeremy M Berg, John L Tymoczko, Lubert Stryer , Neil D Clarke


each transcript can give rise to several protein isoforms via post translational processing (>300 PTMs)

PROTEOMICS

• proteins are the main biological effector molecules, providing structures, enzyme activities, transport and more

• the next step from determining the genome is to find out the function of the gene products – the proteins

• analysis of protein products complements genomics & transcriptomics

“At the end of the day, proteins, not genes, are the business end of biology”

identify every protein present in a cell, tissue, biofluid or organism

Global Proteomics

Catalogue of proteins

most practical for simple organisms eg yeast, prokaryotes

• quantitativeprotein abundance

• qualitativepost translational modifications

phosphorylation, glycosylation

• subcellular compartments/organellesnuclei, plasma membrane, mitochondria

• functionalcomplexes of interacting proteins

Targeted (Sub)Proteomics

Mass Spectrometry

There are many Proteomic Approaches using many different technologies

Protein Chips

Protein arrays on slides

(protein spots, tissue sections)

Liquid Chromatography

Peptides/Proteins1D/2D

Labels/label free

Gels

Proteins1D/2D gels

stains/labels

Electrospray ionization (ESI)

John B Fenn

Matrix-assisted laser desorption/ionization (MALDI)

Koichi Tanaka

"for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules"

The Nobel Prize in Chemistry 2002

Applications of mass spectrometry in protein analysis include

Protein identification peptide mass fingerprintingTandem MSde novo sequence

Recombinant protein evaluationconfirm identityengineered mutations, sequence changescleavages or other modificationsassess homogeneity

Identification of modificationsacetylationoxidationglycosylationphosphorylation

….anything that causes a change in mass….

Proteomic Workflow2D gel/MS

Protein separation

Mass spectrometric analysis

Database interrogation

Analysis and protein spot selection

Processing and digestion to peptides

Protein identification

Protein separation2-dimensional gel electrophoresis

1st dimension Separation by charge(isoelectric focussing) pH 3 pH 10

pI

2nd dimension Separation by molecular weight

(SDS-PAGE) kDa

2D gel electrophoresis equipment1st dimension IEF

various lengths 5 - 24 cm

wide range pH 3-11

narrow/zoom range pH 4-5

loading methods in-gel rehydration cup, paper bridge

2-D gel electrophoresis equipment 2nd dimension SDS-PAGE

various lengths

linear / gradient

reducing / non-reducing

Multi-gel runnersincrease reproducibilityincrease throughput

Protein detection and image capture

post-gel staining

colloidal coomassie bluesilverSYPRO ruby, Deep Purple, Flamingo

pre-gel sample labelling 35S-methionineCy3, Cy5, Cy2 (DiGE)

Pro-Q Diamond – phosphoproteinsPro-Q Emerald – glycoproteinsPro-Q Amber – transmembrane proteins (1D gels)

Example 2D gelE. coli cell extract

Soo Jin Saa100 µg, Silver stained, mini-formatpH 4-7 IPG strip, 12.5% PAGE

100

37

25

150

75

50

20

250

Resolution – how many proteins?

Depends on the separation lengths of gelsie size of IPG strip and 2nd dimension gel

Mini-gels (7 x 7cm) – a few hundred

Midi-gels (18 x 20 cm) – ~1-2,000

Large format (24 x 20 cm) – up to10,000

each spot is adifferent protein

spot intensity isproportional to the amount of protein

Comparison of gel stains

ColloidalCoomassie Blue

10-50 ng/mm2

SYPRO ruby

~ 1 ng/mm2

Silver

0.5 ng/mm2

Proteomic Workflow 2D gel/MS

Protein separation






Analysis and spot selection

Image analysis software

PDQuest (BioRad)

DeCyder (GE Healthcare)

Same Spots (Nonlinear Dynamics)

Image capture

Spot detection

Spot matching across gel set

Statistical evaluations

Find differences in spot patterns (protein expression changes) between samples using image analysis software

separate proteins and compare different samples

2D gel electrophoresis separates proteins in two different dimensions – pH and size

differences in spot intensity = protein expression changes

normal cells diseased cells

Figure courtesy of Dr Rob Layfield, School of Life Sciences

normal cells diseased cells

You found some changes what are the actual proteins?

separate proteins and compare different samples

Proteomic Workflow 2D gel/MS

Protein separation






Gel spot excision and processing

Pick individual spots

into 96-well

microtitre plates

Destain

Digest (trypsin)

Peptide extraction

Identify proteins using Mass Spectrometry

MALDI-ToF Q-ToF2 (plus capillary/nano flow HPLC)

gel versus off-gel

limitations of 2D gels

Some classes of proteins are difficult to obtain on 2D gels

basic / acidic proteinslarge / small proteinsmembrane proteins

Low throughput / difficult to automate

advantages of 2D gels

allow examination of modifications of intact proteinsrelatively low cost equipmenteasy to understand/interpret

Another approach : off-gel high throughput LC-MSMS

Mass Spectrometry

Many Proteomic Approaches using many different technologies

Protein Chips

Protein arrays on slides

(protein spots, tissue sections)

Liquid Chromatography

Peptides/Proteins1D/2D

Labels/label free

Gels

Proteins1D/2D gels

stains/labels

Proteomic Workflow high throughput LC-MSMS

(bottom up)

Digestion of complex protein sample



Protein identification (large numbers)

Quantitationtagging, non-tagging approaches

Peptide separationHigh resolution HPLC

(often multidimensional)

Combines the HPLC separation of peptides with the detection and analytical power of tandem MS

LC-MSMS

Sample

MASSANALYSER

DETECTORESI

IONISATION SOURCE

MudPIT (Multi-dimensional protein identification technology)

Separate complex mixtures of peptides using

multi-dimensional HPLC

1st dimension – strong cation exchange

2nd dimension – reversed phase

Analyse using tandem mass spectrometry

LC-MSMSCombines the HPLC separation of peptides with the

detection and analytical power of tandem MS

Figure courtesy of proteabio.com

multidimensional chromatography LC-MSMS

From Vanderbilt University Medical Centre

high throughput LC-MSMS(shotgun)

2014 first draft maps of the human proteome published:

high resolution mass spectrometry

29 May 2014

Kim et al74 authors, led by Pandey lab in John’s Hopkins University, Baltimore, USA & Institute of Bioinformatics, Bangalore India)

•MS data on 30 normal human tissues and primary cell cultures•identified proteins encoded by 17,294 genes (~84% of predicted ORFs)•found >190 new proteins not predicted from genome sequence•Human Proteome Map http://www.humanproteomemap.org/

draft maps of the human proteome

Wilhelm et al22 authors, led by Kuster lab in Technische Universität München, Germany)

•combined already existing MS data (tissues, cell lines, body fluids, affinity purifications) with their own MS data (altogether 60 human tissues, 13 body fluids, and 147 cancer cell lines)•evidence for over 18,000 proteins (~92% of predicted proteins)•found >400 translated long, intergenic non-coding RNAs (lincRNAs)•ProteomicsDB https://www.proteomicsdb.org/

http://www.humanproteomemap.org/

https://www.proteomicsdb.org/

advantages of LC-MSMSvery high thoughput

identify large numbers of proteinsachieve more analytical runs/replicates

disadvantages of LC-MSMSdigestion increases the complexity of the sample

lose the connection between peptides and intact proteinscomplex analysis procedures/software need more bioinformatics assistance

Often used as complementary approaches, or in combination

gel versus off-gel

• gel based and high throughput off-gel experiments generate huge quantities of data in the form of long lists of proteins

• how can these data be placed in the biological context – what does the data tell you?

• this is a huge challenge

• the need is to establish what the proteins do, what other proteins they interact with and work out why they have changed - in order to obtain molecular insights into the process in question

What next?

Specialised software tools

‒ network analysis toolsIngenuity Systems IPA : pathway & networkanalysis of complex 'omics data

Figure from Rathbone, Liddell & Campbell (2013) Cellular Reprogramming 15:269

‒ text mining tools to help establish the function(s) of each proteinDAVID : functional annotation tools to help understand

biological meaning behind large list of proteins/genes

formulate a testable hypothesis to drive the research forward

Cytoscape : platform for visualizing complex networks and integrating ‘omics data

STRING : database and web resource for known and predicted protein-protein interactions

Challenges in proteomics : Complexity

Many different molecules that are expressed at different

‒ levels

‒ times

‒ places

‒ PTMs


Range of sizes

•from ten to several hundred amino acids

•less than 10 kDa to 1 million kDa

Different forms

•post translational modifications hugely increases the number of different molecules

Chemical composition

•very different physicochemical characteristics

•when extracted from the cell they require different conditions to maintain solubility/stability etc

•large challenges in sample handling


Diversity of sequences (number of genes/ORFs)

arabidopsis ~28,000 (the smallest plant genome)human > 22,000worm ~ 19,000yeast ~ 6,000E.coli ~ 5,000 (fairly typical for bacteria)

Huge number of proteins

estimates of the size of the proteome varies widely

for human cells it varies from 100,000 to 2 million!

Challenges in Proteomics

Dynamic range

Don’t see the lower abundance proteins in complex mixtures

Anderson NL, Anderson NG The human plasma proteome: history, character, and diagnostic prospects

Molecular and Cellular Proteomics 2002 1:845-867

Proteins measured clinically in plasma span > 10 orders of magnitude in abundance

1010 Really Is Wide Dynamic Range(Here on a linear scale)

2D gels only ~2-3 orders of proteins detected only the most abundant proteins

Mass spectrometers detection range of ~ 3 to 5 orders (recently maybe 6)

Dynamic range is a problem in proteomics since this one feature alone makes it impossible to analyse every molecular species present in a proteome

What is detectable?

Reduce the complexity and dynamic range

Fractionation techniques include remove abundant proteinsdifferent cellular compartmentsdifferential protein solubilitysequential chromatography (2D, 3D)affinity purification

How to overcome the dynamic range and detect proteins of lower abundance?

Fractionation : Remove abundant proteins12 proteins in plasma comprise ~ 96% of the protein mass

Figure courtesy of Beckman Coulter

Immunodepletion of 6 high abundance proteins from human serum

1 - crude serum

2 – flow through fractiondepleted of high

abundance proteins

3 - bound fraction

M 1 2 3

Figure courtesy Agilent Technologies

uses unbiased global screening technologies to analyse very complex samples

a proteome is dynamic and can vary depending on physical conditions, cell cycle, environment,

health/disease etc

a proteome is a “snapshot “of protein expression/modification by specific cells/tissues, under

particular conditions

identifies protein targets for further investigation after validation

Proteomics

Utube videoHow to run great 2D Gels (BioRad)

http://www.youtube.com/watch?v=7R_R6mbqvFk

Nat Rev Mol Cell Biol 2004 5(9):699

J Proteomics 2011 Sep 6;74(10):1842

Matrixscience MASCOT help pages http://www.matrixscience.com

MASCOT video tutorialshttp://view6.workcast.net/?

pak=3003276531895477&cpak=7053452047055213

References and other sources of information

Journal of Proteomics 2011 74:1829

http://www.youtube.com/watch?v=7R_R6mbqvFk

http://www.matrixscience.com/



http://view6.workcast.net/?pak=3003276531895477&cpak=7053452047055213

http://view6.workcast.net/?pak=3003276531895477&cpak=7053452047055213

References and sources of further information

WEBSITES

A Mass Spectrometry and Biotechnology Resourcehttp://www.ionsource.comMS Protein Identification Tutorial - especially De Novo Peptide Sequencing Tutorial ExPASy : Bioinformatics Resource Portalhttp://www.expasy.org/proteomicshttp://world-2dpage.expasy.org/swiss-2dpage/

Human Proteome Organisation (HUPO)http://www.hupo.org

The Fixing Proteomics Campaignhttp://www.fixingproteomics.org/ Guide to mass spectrometryhttp://masspec.scripps.edu/mshistory/whatisms_details.php#Basics

Nat Rev Mol Cell Biol 2010 11:789

Nature Biotech 2010 28:695

Annu Rev Biochem 2011 80:239

http://www.ionsource.com/

linkshttp://view6.workcast.net/?pak=3003276531895477&cpak=7053452047055213 MASCOT video tutorials

http://www.ionsource.com/ Mass Spectrometry and Biotechnology Resource – lots of useful info – tutorials on de novo sequencing etc

http://www.swissproteomicsociety.org/digest Swiss Proteomics Society. The “digest” provides a consolidated selection of articles published in all scientific

publications that are pertinent to proteomics – finds all the interesting and relevant papers for you!

http://proteome.nih.gov proteomics special interest group at NIH, includes archived videocasts of research seminars

http://ca.expasy.org/tools/ proteome informatics tools e.g. peptidemass predicted digestion fragment tool

http://www.bspr.org/ British Society for Proteome Research

http://www.bmss.org.uk/ British Mass Spectrometry society http://www.plasmaproteome.org/ The Plasma Proteome Institute in Washington D.C.

http://www.unimod.org/ Unimod : protein modifications for mass spectrometry

http://www.hupo.org/

http://www.spectroscopynow.com/coi/cda/home.cda?chId=0

http://www.abrf.org/index.cfm/group.show/Proteomics.34.htm

introduction to proteomics susan liddell university of nottingham [email protected] pgt...

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