Recent advances in MS-based Proteomics

Proteomic Analyses by Mass Spectrometry Iris FinkemeierMPI Plant Breeding Research 14 January 2014finkemeier@mpipz.mpg.deThis talkConcepts of MS-based proteomics The three main applications:Description & identificationQuantitationInteraction2Whats a proteome?3Genes to proteinsGene Transcript Protein TranscriptionTranslationTRANSCRIPTOMEPROTEOME(mRNA)The proteome is the entire set of proteins expressed by a genome, cell, tissue or organism at a certain time. 5Whats proteomics?6 ProteomicsThe goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations such as disease or drug treatment.

From: N. Leigh Anderson and Norman G. Anderson: Proteome and proteomics: New technologies,new concepts, and new words, ELECTROPHORESIS 19, 1853 1861 (1998) Lack of correlation between transcript and protein

AGPaseFd-GOGATProteomics at the sweet spot9

Hoog & Mann, Annu Rev Genom Human Genet (2004)e.g. 20,000 genesSpliceforms (~ 5x increased complexity)Post-translational modifications(> 200 PTMs known, 10x increased complexity)How complex is complex?Human genome: 20.300 predicted protein coding genes230 cell types, body fluidsSpliceformsPost-translational processing and modificationsInteractions and protein complexesHuman proteome project (launched 2011)!10Protein coverage & technological innovation

MascotShotgun proteomicsLTQ-FTOrbitrapSynaptAhrens et al., Nat Rev Mol Cell Biol (2010)11Brenfaller et al., Nature (2008)Arabidopsis proteome> 13.000 proteins identified11Proteins -reminder of the basics-12-Amino acidsThe 20 amino acids specified by the genetic code have the general formula:NH2H C RCOOHL-alanine

The size, polarity and hydrogen bonding properties of the side chain (R) attached to the -carbon atom confer unique properties on each amino acid.13Aliphatic non-polar side chains

The amino acids in this group have side chains with no affinity for water.These amino acids tend to be found in the hydrophobic core of water soluble proteins, or in contact with lipids in membrane proteins.14Uncharged polar side chainsThe electronegative oxygen atom confers a degree of polarity on the side chains of asparagine, glutamine, serine and threonine, allowing the formation of hydrogen bonds.

The sulphur atom is only weakly electronegative and the cysteine side chain does not form hydrogen bonds.These amino acids will often be found on the surface of a soluble protein, but their weak polarity means that they can also be easily stabilised in the interior of a folded protein.15Charged polar side chainsSome side chains ionise at normal physiological pH values.

H+Charged side chains only occur in hydrophobic regions if they can form an ion pair with an oppositely charged side chain.Amino acids with charged side chains are often found on the surfaces of soluble proteins.16Aromatic side chainsThe aromatic side chains are predominantly non-polar, but the OH group in tyrosine and the NH group in tryptophan allow these side chains to form H-bonds.

Phenylalanine has the most non-polar side chain of all the amino acids and hence the strongest preference for a non-aqueous environment.This preference can be quantified and it decreases in the order:Phe > Met > Ile > Leu > Val > Cys > Trp > Ala > Thr > Gly > Ser > Pro > Tyr > His > Gln > Asn > Glu > Lys > Asp > Arg.17The peptide bondCondensation of two amino acids results in a peptide bond:NH2 CH C OHR1ONH2 CH C OHR2ONH2 CH CR1ON CH C OHR2OH+The condensation process is energy consuming (~160 kJ mol-1) and it can be repeated many times to produce polypeptides containing hundreds of amino acids.The peptide bond has partial double bond character, preventing free rotation about the carbon-nitrogen axis:CNHOCC1819

Average length of proteins)201. Classical gel-based proteomicsUp to 1000 proteins can be reproducibly resolvedon a single gel

3 4 5 6 7 8 9 10 pI944366302014kDa2-D gel electrophoresis

21The advent of precast IEF gels with IPG means that we can no run highly reproducible first dimensionsThis is a typical 2D gel showing 800 ug of total protein from an Arabidopsis cell suspension culture stained with colloidal coomassie blueYou can see that up to 1000 proteins can be resolved on a single gel and many more can be seen if you first fractionate your protein sample and runt the fractions on separate gels.1. Isoelectric focusingCOOHCOO- + H+

NH3+NH2 + H+10Anode (+)Cathode (-)+-3pH gradient2D gel electrophoresisAlkaline pHAcidic pH22Generally 2D gels use isoelectric focusing in the first dimension and conventional SDS-PAGE in the second.IEF takes advantage of the fact that the ionisation of side groups on proteins is dependent upon pH.Thus for the carboxylic acid group shown here, at high pH it will tend to dissociate and will be negatively charged and at low pH the equilibrium will be shifted towards the protonated uncharged form.Different groups have different dissociation constants and their ionisation state will vary accordingly at any given pHA particular protein will have a unique isoelectric point, the pH at which it will have an equal number of +ve and ve charged groups and will therefore carry no net chargeWe can separate proteins according to their pI by electrophoresing them through a gel that contains a pH gradient.Below its pI, a protein will be +ve chgd and will migrate to the cathode. Above its pI, -ve and to anode.Proteins will therefore congregate at the pH that represents their pI where they carry no net charge.2D gel electrophoresis2. SDS-polyacrylamide gel electrophoresis---Anode (+)Cathode (-)polyacrylamide gel231st Dimension (IEF)Separation by chargeSeparation by size2nd DimensionSDS-PAGEanalysis of 2D-gels3 replicate gels for the control and each treatment were runGels were imaged using a CCD-based imaging systemImages were analysed using PDQuest software.

1. Spot detection& quantitation

2. Spot matching

3. Data AnalysisStatistical testst-testMann-Witney signed rank testWilcoxon paired sample testPartial least squares test

Proteomics analysis of protein complexes?

26

Blue native PAGE of mitochondrial respiratory complexes

1-Dimension28WorkflowMembrane isolationSolubilization of protein complexesBN-PAGECell fractionationDenaturation of protein complexesSDS-PAGE2829Dodecyl--D-maltoside

glugalxylDigitoninDetergents29301.1 mM -DM

or

4.5 mM Digitonin4.5-9 mM -DM or 9-18 mM DigitoninDecreasing molecular mass of protein complexesIncreasing concentration of detergent micellesEffect of detergent concentration on membrane solubilization

Blue native PAGE of mitochondrial respiratory complexes

1-Dimension32Comparison of different staining methods after BN/SDS-PAGE : one gel, three stains

Fluorescence

Coomassie

Silver33CyDye labeling of membrane protein complexes

633 nm

532 nm

Cy5

Cy3

Cy2

EmEx488 nm342D BN/SDS-DIGE of plastid membrane complexes

condition I versus condition IIBN-PAGESDS-PAGE

condition IBN-PAGESDS-PAGE

condition IIBN-PAGESDS-PAGE+=35Protein identification by mass spectrometry!Gel-based vs. gel-free proteomics361D / 2D geldigestN-terminal sequencing,mass spectrometryImmunodetectionSpot excisionLC-MS/MSDatabase search (genome!!!)

Ponceau S

AOX

Protein IDProtein ID

Gel-based vs. gel-free proteomics371D / 2D geldigestN-terminal sequencing,mass spectrometryImmunodetectionSpot excisionLC-MS/MSDatabase search

Ponceau S

AOX

Protein IDProtein ID

1 gel1 blot1 protein ID1 sample per spot/protein ID> 1000 protein IDsper sample1 sample1000 samples!1000 hybrid.,1000 antibodies!38Identification of proteins by mass spectrometry analysis

Bottom-up proteomics(Top-down proteomics)

A very short LC-MS/MS introduction40

digestLiquid chromatography (LC)Tandem mass spectrometry (MS/MS)Coupling of HPLC and MS

SampleHPLC(separation/UV detection)MSTIC = total ion countBasic Components of a Mass Spectrometerion source mass analyzer detector

generate ions ion separation ion analysis

ESIMALDIQuadrupolIontrapsTOF (Time of Flight)Used for Peptides and ProteinsWhat LC-MS/MS doesmeasuremasses

MS1MS2fragmentationECCHGDIIECADDRECCHGDIIECADDRECCHGDIIECADDRECCHGDIIECADDRIdentifying Proteins by peptide mass fingerprinting (PMF)2. Translate3. Predict trypsin cleavage sitesGenomic DNA1. Identify genesPredicting tryptic-fragment masses4. Calculate theoretical masses of tryptic fragments 813.432825.501869.482890.975969.5441025.5671069.5541082.0421113.5651141.5962249.082

813.432825.501869.482890.975969.5441025.5671069.5541082.0421113.5651141.5962249.082THEORETICALOBSERVED45

Example: BSA46

Trypsin cleavage site prediction(web.expasy.org/peptide cutter)47

48

How can Peptides and Proteins Be Ionized?Elektrospray-Ionisation (ESI) John B. Fenn

Matrix-Assisted Laser Desorption/Ionization (MALDI)

Koichi TanakaFranz Hillenkamp Michael Karas

50

51

MS/MS Provides Information About Peptide Sequences

SupportQuadrupole1st MassQuadrupolecollisionchamber2nd MassQuadrupoleQ0 Q1 Q2 Q3 Detectorm/z = constant fragmentation m/z scanning 53Important features of a mass spectrometer

Resolution Accurate detection Scan speed Sensitivity Dynamic range

54Important features of a mass spectrometer

Resolution Accurate detection Scan speed Sensitivity Dynamic range

55

Peptide Fragmentation (MS2, tandem mass spectrum)

MS/MS spectrum interpretation & database search

A simple chromatogramseparate

What a real, complex sample looks like

What a real, complex sample looks like

retention timeWhat a real, complex sample looks like

~ 100 putative peptides within 1 minCoverage?How many proteins can you detect?In a 4 h run on the newest generation instrument:25.000 peptides4.000 proteins

Sample requirement: 4 g peptide sample

Sample preparation time: 6h plus digest time6263Acquired peptide masses can be analysed by MASCOT peptide Mass Fingerprint analysis

66

67

This talkConcepts of MS-based proteomics The three main applications:Description & identificationQuantitationInteraction68Protein atlas:mapping complete proteomes69The Arabidopsis proteome atlas 1.070

~ 50% coverageFinding new genes or gene models71

Brenfaller et al., Nature (2008)Finding new genes or gene models72

Brenfaller et al., Nature (2008)Finding new genes or gene models73

Brenfaller et al., Nature (2008)Finding new genes or gene models74

Brenfaller et al., Nature (2008)Exploring sub-proteomes at fine scale75

mapping of 2000 root proteins700 specific for single cell typeMost data are fully accessible76

http://gator.masc-proteomics.org/Mapping post-translational modifications77digestLC-MS/MSDatabase search

Phospho-protein ID

PPPPaffinity-based enrichmentMapping post-translational modifications78

http://phosphat.mpimp-golm.mpg.de/db.htmlNumber of phosphoproteins: 5.460Total no. of phosphopeptides34.700No. of unique phosphopeptides: 13.205Other MS-accessible PTMs: AcetylationOxidationNitrosylationUbiquitinationGlycosylationThis talkConcepts of MS-based proteomics The three challenges and potentials:Description & identificationQuantitationInteraction7980

+-treatmentProteinXFor 100 proteins:100 antibodiesMany gels, blots, workhours, Sometimes there is no antibody!Western-blots are quantitative.81massintensity

1 : 12 : 1

MS is not inherently quantitativeMS is not inherently quantitative82

digestLC-MS/MS

Electrospray ionizationPeptides havedifferent physicochemical propertiesdifferent flyability in MS

Intensity of different peptides is not a direct measure of their abundance!massintensity

Solutions of quantitative proteomicsComputational solutions (on protein level)Label-free analysis, iBAQIntermediate accuracy, stable conditions required

Isotopic labeling 83Stable isotopic labeling84m/zintensity

two sampleswith isotopically labeled proteins (e.g. 13C, 15N)lightheavyratio calculationand quantification1:31:3Quantifying peptide abundance by comparison of the intensity of MS1 spectrumProteinpeptideratio H/LProtein 111.04Protein 1250.6

Protein 1, peptide 1

Protein 1, peptide 2lightheavylightheavyIsotopic labelingAbsolute, add known amount of:Synthetic AQUA peptidesPurified isotopically labelled proteins

Relative, compare between samples:Metabolic labelingChemical labeling86SILAC-Based Proteomics Analysis Biological Question:

How does the expression level of cellular proteins change during differentiation?

Addressing this Question with SILAC:

Differential Isotopic labeling of the cells in differentiated and non-differentiated stage. Separate and analyze the proteins by MS and MS/MS. SILAC: Stabile Isotope Labeling with Amino Acids in Cell Culture Isotope Labeled Amino Acids Commonly usedin SILAC Experiments

Lysine-0 Lysine- 4 Lysine-6 Lysine-8

Arginine-0 Arginine-4 Arginine-6 Arginine-10

Metabolic labeling: SILAC89m/zintensityratio calculationand quantificationSILAC:Stable Isotope Labeling with Amino Acids in Cell Culture

Mix lysate or cells 1:1digestLC-MS/MSArg, Lys lightArg, Lys heavyMetabolic labeling in plants90m/zintensity

ratio calculationand quantification

14N15NChemical labeling: dimethylation as exampleaddition of two methyl groups to all - and -amino groups

Boersema et al., Nature Protocols, 2009.Stable isotope dimethyl labeling

Boersema et al., Nature Protocols, 2009.Stable isotope dimethyl labeling

Boersema et al., Nature Protocols, 2009.Labeling strategies: an overview94

Bantscheff M. et al., Anal Bioanal Chem (2007)Yeast as an example953824 proteins identified and quantified67% ORF coverage80% expressed, 800 dubious ORFs~82% real coverage

Around 75 samples and 300h measuring time!

pheromone responseRapid technological progress96

2011: Thermo: Q-Exactive OrbitrapWaters: Synapt G2

leads to new depths in coverage.97

Nagaraj et al., MCP, 201285-90% coverage per 4h run!Proteome coverage in yeast98

Protein concentrationNumber of protenis

proteins identified& quantifiedall proteins in sampleLarger proteome, less coverage99

Bantscheff M et al., Anal Bioanal Chem (2007) 389:10171031HeLa proteome (10000 proteins) is largely covered but with lots of samples and measuring time.Faster and reliable alternatives for selected proteins?Problem like in the early days of microarraysIncomplete coveragelow abundant transcripts/proteins are hard to quantify reliably

What would the molecular biologists do?quantitative real-time PCR!100The qPCR in proteomics: SRM and MRMSRM - selected reaction monitoringMRM - multiple reaction monitoring

Targeted proteomicsNature method of the year 2012!101

Why SRM is appealing102

Selectivity!

Database search

LC-MS/MSSRM: the basic idea103

Gilette & Carr, Nature Methods, 2013SRM: the basic idea104

Gilette & Carr, Nature Methods, 2013Targeted proteomicsadvantages:fastcheaper & more robust instrumentshighly sensitivepromising for clinical applications!

disadvantages:optimisation & standards neededlimited to a max. of 100 proteins per run105This talkConcepts of MS-based proteomics The three challenges and potentials:Description & identificationQuantitationInteraction106