Protein Phosphorylationand

Sample Prep

Johan ÖhmanSenior ScientistGE Healthcare Bio-Sciences AB

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Outline

Introduction

Case Study 1 pervanadate treated CHO cells

Case Study 2 phosphoproteomics

Case Study 3 drug treatment of cancer cells

Summary

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Background protein phosphorylation

Over 200 PTM’s exist but only a few are reversible.

Phosphorylation is vital for many cellular functions:

•signal transduction•cell differentiation•cell cycle control•”on-off” switch

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Background protein phosphorylation

~15% of total proteins (some cellular state)

Differences between Ser/Thr and Tyrphosphorylations.

Relative abundance:Ser Thr Tyr

1000 / 100 / 1

N

O

OP

O

O O

N

O

OP

O

O O

N

O

OPO

O

O

Tyrosine-phosphate

Threonine-/Serine-phosphate

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• apoptosis

• cell cycle / DNA repair

• kinases

• signaling

• oncogenes / tumor suppressors

• phospho proteomics

• interaction proteome

• localization and dynamics

• cancer

• diabetes / obesity

• immunology

• angiogenes

• pathogenes

Cellular processes Therapeutic

Other

Phosphorylation - research areas

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Phosphorylation - detection

• PAGE / 2-D PAGE

• Immunoblot

• Direct staining

• DIGE

+separation power, sensitivity,

overview of complex sample

-poor protein representation, limited

Mw range, membrane proteins..

• MS after proteolytic digestion

• MALDI-ToF/ToF

• LC-MS/MS

+precise location of phosphorylation

site(s), identification, membrane proteins, automation..

-complex sample mixtures, incomplete

fragmentation MS/MS

Techniques

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SampleDownstream application

Sample preparation Analysis

Sample preparationWorkflow

Sample specific

Coreprocedure

Downstream specific

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Sample prep - improves the quality

Minimize

• degradation

• sample loss

• contamination

• in vitro chemical modifications

… leads to enhanced

• signal for low abundance molecules

• analytical resolution

optimize the quality ofthe sample

maximize the quality ofthe result

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Sample prep - enrichment techniques

Affinity

matrix

Sample mixture

pTyr-antibody

IMACpSer-/pThr-

antibody

Chemical

modification

Immunoprecipitation

Zr4+ Al3+

Fe3+Ga3+

MOAC

TiO2

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Sample prep – workflow phosphoproteins

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Sample prep - phosphoproteins

• Phosphatase inhibitors

• Reduce phosphatase activity

• Obscured (hidden) PO4 groups

• Mapping protein interactions

• Multi-protease approach may be needed (LC-MS/MS)

• Reduce ”background” by using good controls

• Low abundance

Amount material needed:

100-200 fmol peptide in LC-MS/MS

10-200 million cells, 1-20 ml cell extract

Important considerations

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Mag SepharoseTM

Attractive sample preparation made easy

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Products for enrichment of proteins and phosphopeptides

Magnetic beads Additional material

NHS Mag Sepharose™Protein A Mag SepharoseProtein G Mag SepharoseTiO2 Mag SepharoseMagRack 6

Low µg scaleVariable amount of beadsVariable sample volume

Specific Ligands, e.g.:AntibodiesOther Binders

Enzyme inhibitors, e.g.:Protease inhibitorsPhosphatase inhibitors

Extraction and immunoprecipitation buffers

Your Sample – Cells or tissue

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Enrichment of phosphoproteins and peptides

Case study 1:Finding low abundant tyrosine phosphoproteins in CHO cells

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Key sample preparation issues for phosphoprotein and phosphopeptide analysis

Coverage of the phosphoproteome: Selective enrichment of phosphoproteins and –peptides

Alteration of the phosphoproteome:Stop in vitro dephosphorylation

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Sample prep - enrichment techniques

Affinity

matrix

Sample mixture

pTyr-antibody

IMACpSer-/pThr-

antibody

Chemical

modificationImmunoprecipitation

Zr4+ Al3+

Fe3+Ga3+

MOAC

TiO2

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Sample prep – workflow phosphoproteins

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Biological systemCHO cellsTreatment with pervanadate (H2O2 + vanadate), known to introduce a decrease in tyrosine phosphorylation

Tools and methodsPhospo tyrosine specific antibody, PY20 Protein G Mag Sepharose™ beads TrypsinMass spectrometry, LC-MS/MS

Changes in tyrosine phoshorylationin CHO cells upon pervanadate treatment

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Enrichment of tyrosine phosphorylated proteins in CHO cells

Trypsin digestion

™

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• 76 potential tyrosine phosphoproteins identifiedSingle analysis , simple MS method..

• Enrichment with Protein G Mag Sepharose™ immobilized with an anti-pTyr-antibody offers a sensitive and efficient capture of the low abundant pTyr proteins

Changes in tyrosine phoshorylationin CHO cells upon pervanadate treatment

Oxidative stress indicated !

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Enrichment of phosphopeptides

Case Study 2:Phosphopeptides in a leukemia cancer cell line enriched by TiO2

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Sample prep – workflow phosphoproteins

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Enrichment and identification of phosphopeptides in cancer cells

Workflow for analysis of phosphopeptides from leukemia cell line

LC-MS/MS analysis and peptide identification

TiO2 Mag Sepharose ™protocol

Desalting of peptides (C-18)

Trypsin digestion of proteins in cell lysate

Cell lysis

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NoY1601yLEESDEDDLF183 kDa protein/DNA topoisomerase 2TOP2AIPI00178667

YesS214NKPGPNIEsGNEDDDASFKEukaryotic translation initiation factor 5BEIF5BIPI00299254

YesS218IYHLPDAEsDEDEDFKEQTRSeptin-2Septin-2IPI00014177

YesS136DKsPVREPIDNLTPEERIsoform 1 of RNA-binding protein 39RMB39IPI00163505

No, sequence specific for isoform P

S546/S547AssLNVLNVGGKIsoform P of Kinesin light chain 1KLC1IPI00337465

YesY849LCDFGSASHVADNDITPyLVSRSerine/threonine-protein kinase PRP4 homolog

PRPF4BIPI00013721

YesY15IGEGTyGVVYKCell division protein kinase 3CDK3IPI00023503

Yes, by MAPKAPK2 and MAPKAPK3.

S179SQsDCGELGDFRProtein kinase substrate CapZIPRCSD1IPI00017659

No, S139 knownY137 (or S139)GLLyDSDEEDEERPARDNA replication licensing factor MCM2MCM2IPI00184330

YesS188RDsFDDRGPSLNPVLDYDHGSRMatrin-3MATR3IPI00017297

NoS11SSsFGNFDRSAM-domain protein SAMSN-1SAMSN1IPI00185526

YesS82QLsSGVSEIRHeat-shock protein beta-1HSPB1IPI00025512

Yes, by CK2S366TKFAsDDEHDEHDENGATGPVKRLupus La proteinSSBIPI00009032

Yes, by CK2S366FAsDDEHDEHDENGATGPVKRLupus La proteinSSBIPI00009032

Yes, for all 3S230/S231/S232 sssPAPADIAQTVQEDLRRas GTPase-activating protein-binding protein 1

G3BP1IPI00012442

SwissProt3

SitePhosphopeptide sequence2

Protein nameGene nameAccession number1

Identified proteins & phosphopeptide sequences

No phosphopeptides identified before enrichment !

Enriched sample identified 15 phosphopeptides

TiO2 binds to both Ser/Thr and Tyr phosphopeptides

* Data were kindly provided by Sara Lind, Lu Lu, Lioudmila Elfineh (PhD) and Prof. Ulf Pettersson, Department of Genetics and Pathology, Uppsala University; and Konstantin Artemenko (PhD) and Prof. Roman Zubarev, Department of Cell and Molecular Biology, Molecular Biometry Group, Uppsala University.

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Enrichment of phosphoproteins and peptides

Case Study 3:Changes in tyrosine phosphorylation in cancer cells upon drug treatment

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Sample prep – workflow phosphoproteins

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Low abundant proteins are often biologically important but difficult to detect

Regulatory proteins are often present in low amounts and only for a short time.

Labeling or staining will result in very weak or no signal due to limits in detection or sensitivity of label or stain

Challenges for low abundant proteins

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Use more sensitive stains or labels

Enrich the protein or group of proteins

Use antibody based detection and amplify the signal

Perform selective labeling

Remove high abundant proteins

Over express your protein of interest

How can we improve the possibility for detection of low abundant proteins?

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Select a separation method for optimal resolution of protein of interest

•1-D SDS-PAGE

•2-D electrophoresis

•1-D Western

•2-D Western

•IEF gels

•pH interval•Gel size•Acryl amide content

Method:

Parameters:

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high (pg)

low (ng)

Coomassie™Blue

SilverRadio

isotopes

Deep Purple™

Sypro™ Ruby

CyDye™

ECL Plex™

Quantification range

sensitivity

31Pixel position

Sign

al in

tens

ity(c

ount

s)

Lane 22 Lane 23 Lane 24 Lane 25

What sets limit of detection?

Signal intensity Background Variation in background (noise)

Signal: noise ratio ≥ 3 is limit of detection

Amount of sample

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Biological systemK562 chronic myeloid leukemia cells

Treatment with imatinib (Gleevec), known to introduce a decrease in tyrosine phosphorylation

QuestionsCan we detect and analyze changes in the very low abundant tyrosine phosphorylated proteins?

Can we identify the proteins that are differentially regulated?

Collaboration with Uppsala University, Uppsala, Sweden: Dr. Sara Lind et al, Rudbeck laboratory

Changes in tyrosine phoshorylationin cancer cells upon drug treatment

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Simplified DIGE experiment

CyDye™ pre-labeling 2-D Electrophoresis Imaging

Control (Cy™2)

Imatinib treated (Cy3)

Total protein samples

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No significant change in protein abundances in total protein samples

Immobiline™ drystrip: pH 4-7, 7 cmSDS-PAGE gel: 4-20 % Tris Glycine, 8 x 7 cm

control

treated

overlay

Data courtesy: Sara Lind, Rudbeck laboratory, Uppsala , Sweden

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Use more sensitive stains or labels

Enrich the protein or group of proteins

Use antibody based detection and amplify the signal

Perform selective labeling

Remove high abundant proteins

Over express your protein of interest

How can we improve the possibility for detection of low abundant proteins?

36

Enrichment of proteins and peptides using Mag Sepharose™ beads

NHS Mag SepharoseProtein A Mag SepharoseProtein G Mag SepharoseTiO2 Mag Sepharose (peptides)

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Analysis of low abundant phosphoproteins

Tools and methodsPhospho tyrosine specific antibody, 4G10

Protein G Mag Sepharose™ beads

2-D fluorescence differential gel electrophoresis (2-D DIGE)

DeCyder™ 2-D differential analysis software

Mass spectrometry

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Work flow enrichment of pTyr proteins

Phenyl phosphate (phospho tyrosine analogue)

Overnight incubation with K562 cell lysateat +4ºC

Desalting, concentration andexchange into DIGE labeling bufferVivaspin ultracentrifugation columns (MWCO 5 kDa)

Anti phospho tyrosine antibody, 4G10

Protein G Mag Sepharose™

VivaSpin™

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Down regulation detected by DIGE after enrichment

Protein G Mag Sepharose™ + anti pTyr 4G10Immobiline™ drystrip: pH 4-7, 7 cmSDS-PAGE gel: 4-20 % Tris Glycine, 8 x 7 cm

control

treated

overlay

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DIGE results before and after enrichment

Non-enriched Enriched

Immobiline™ drystrip: pH 4-7, 7 cmSDS-PAGE gel: 4-20 % Tris Glycine, 8 x 7 cm

Protein G Mag Sepharose™ + anti pTyr 4G10Immobiline™ drystrip: pH 4-7, 7 cmSDS-PAGE gel: 4-20 % Tris Glycine, 8 x 7 cm

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Many tyrosine phosphoproteins are down regulated upon drug treatment

Control Imatinib treated overlay

DeCyder™ 2-D differential analysis software

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-11.13

ControlImatinibtreated

-5.14

-11.52

Large down regulation

Fold change

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-2.33-11.13gi 4504111Growth factor receptor-bound protein 2 isoform 1

GRB210

-2.39-2.65-n/a-9

-8.81-3.14-n/a-8

-11.56-11.62P6198114-3-3 protein gamma14-3-3 γ7

-9.98-5.14gi 580322514-3-3 protein epsilon 14-3-3 ε6

-1.48-3.09gi 114155148Tropomyosin 3 isoform 5TPM35

-1.95-2.93-n/a4

-2.47-2.64gi 4885153v-crk sarcoma virus CT10 oncogenehomolog (avian)-like

CRKL3

-1.94-2.93-n/a-2

-2.07-2.68-n/a-1

Gel 22Gel 12Protein accession1

Protein full nameProteinSpot no

Ratio control/treated

Protein Identification results

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Control

Drug treated

Total protein pre-labeled with Cy™3Unlabeled total protein

Anti phospho tyrosine primary, 4G10ECL Plex™ Cy 5 secondary

2-D Western blotting showed decrease in tyrosine phosphorylation

Membrane imageCy 3/Cy 5 overlay

2-D electrophoresis

Transfer to membrane

Antibody probing

Cy 3

Cy 5

P

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Enrichment enabled the detection of differences in very low abundant proteins

Tyrosine phosphoproteins were decreased in response to drug treatment

Results from analysis of CyDye™ labeled enriched proteins and Western blotting were in good agreement

It was necessary to enrich the proteins to get protein identity with MS

Conclusions, case study 3

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Summary

Enrichment of low abundant phosphoproteins is necessary for detection and identification

Good sample prep methods are necessary for high quality results

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Thank You for your attention!

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Questions!

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Recently LaunchedSample Prep Products

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Target protein analysis in crude samples –protein activity

Crude sample Conditioning Various assays

Desalting

Concentration

Know your protein activity

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Antibody binding –Fluorescent Western blotting

Relative increase of His-GFP

Quantification of targetprotein relative to an internal, endogenous standard

Multiplexed fluorescent detection for reliable

quantification

t0 t1 t2 t3 t4

RuvB(house-keeping protein)

His- GFP(target protein)

t0 t1 t2 t3 t4

ECL Plex™

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Antibody binding-Fluorescent Western blotting

Primary antibody

Targets on membrane

= Target protein = House-keeping protein(internal standard)

Cy 3 Cy 3

ECL Plex™ secondary antibody CyDye™ Conjugated

Cy™5 Cy 5

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Minimal CyDye™ DIGE fluors

Minimal labeling• 50 µg protein• single label (1-3 %)• ε-amino group of lysine

3 dyes: Cy™ 2, Cy 3, Cy 5• charge matched (+1 charge)• size matched (~450Da) • labeled samples co-migrate• detection limit ~0.25 ng• linear dynamic range: over 4 orders of magnitude

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Target protein analysis in purified sample

Decreasingspecificity

SDS-PAGE

Gel Filtration

Size / apparent molecular weight

UV/VIS spectroscopyUnique fluorescence / absorbance

Western blotting, ELISA etc.

Antibody binding

Depending on type of activity

Protein activity

MSMass

Chemical or MS/MSAmino acid sequence

AssayTarget protein property