Sample preparation and measurement strategies in phosphoproteomics

Boris MačekProteome Center Tübingen

MaxQuant Summer SchoolMartinsried, June 26, 2013

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DNA RNA Protein

Genome Transcriptome Proteome

Protein*

PhosphorylationGlycosylationDisulfide bondsProteolysisAcetylationMethylationSulfationUbiquitinationGPI Anchoretc...

Central „dogma“ of molecular biology

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Figure 3-64 Molecular Biology of the Cell (© Garland Science 2008)

Most (if not all) proteins are modified

Regulatory modifications are dynamic:

• kinase/phosphatase• acetyltransferase/deacetylase• ubiquitin ligase/deubiquinating enzyme• glyosyltransferase/deglycosidase

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MS-based proteomics

Aebersold R and Mann M. 2003. Nature 422: 198-207 4

Choudhary and Mann. 2010. Nature Rev Mol Cell Biol. 11:427

PTMs analyzed in large scale by MS

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Choudhary and Mann. 2010. Nature Rev Mol Cell Biol. 11:427

Largest phosphoproteomes reported so far

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Gel-free phosphoproteomics workflow

Antibodies:4G10pY20, pY99, pY100

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1st stage of enrichment:Strong Cation Exchange (SCX) chromatography

Beausoleil et al. 2004. PNAS 101(33): 12130-12135 8

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1st stage of enrichment:Strong Cation Exchange (SCX) chromatography

2nd stage of enrichment:IMAC or TiO2 chromatography

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Unmodified peptide(change in protein level)

Modified peptide(change in modification level)

No change

Upregulation

Downregulation

Quantification of PTMs

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Unmodified peptide(change in protein level)

Modified peptide(change in modification level)

No change

Downregulation(position?)

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Quantification of PTMs

Intracellular signaling networks:EGFR Signaling Pathway

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Global phosphorylation dynamics

6600 phosphorylation-sites from more than 2000 proteinspS (87%)/pT (12%)/pY (1.5%)

→ www.phosida.com

Less than 15% regulated by EGF treatment

Global understanding of how the cell works - Systems biology modelling of signaling networks

Generic approach – can be applied to study any phosphorylation dependent signal network

Olsen et al., Cell 2006, Volume 127, Issue 3 , p. 635-648

The EGF induced phosphorylation signal spreads to many different protein classes within 20 min of stimulation

Main conclusions:

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• targets of Aurora kinase in S. pombe (with S. Hauf, FMI/MPI)• Koch et al. 2011. Science Signaling 4(179):rs6

• targets of Polo and Fin1 in S. pombe (with I. Hagan, Paterson Institute)

• targets of protein kinase D in human cells (with A. Hausser, Uni Stuttgart) • Franz-Wachtel et al. 2012. MCP

• targets of S/T kinases and phosphatases in model bacteria

Detection of kinase substrate candidates

Control cells Kinase Inactivation

WB WB

Kinase inactivation:

1)By chemical inhibition2)By overexpession of inactive kinase („dominant-negative“ mutant)3)By gene modification (analog-sensitive kinases)4)By gene knockout (only non-essential kinases!)

Adapted from Kettenbach et al. Science Signaling 2011, Vol 4 Issue 179 rs5

Detection of kinase substrates:Kinase inactivation

nanoLC-MS/MS

Lys-12C614N2

Control cells Kinase Inactivation

Combine and lyse

”normal AA” ”heavy AA” (+8Da)

Proteolysis(Lys-C)

SCX

TiO2

GeLC-MS(15 slices)

Lys-13C615N2

Detection of kinase substrates

Figure 15-53, 15-54 Molecular Biology of the Cell (© Garland Science 2008)

Dominant–negative kinase mutants

Example: Receptor Tyrosine Kinases

• overexpression of inactive kinase → suppression of endogenous kinase activity (also called „kinase-dead“ strains)• problem: some endogenous kinase activity remains

Construction of analog-sensitive (as) kinases

• „gatekeeper“ amino acid in the ATP-binding pocket is removed• the ATP-binding pocket can fit an ATP analog or and inhibitor• specific inhibition!

Example 1: Use of analog-sensitive kinases

Detection of Aurora kinase targets in S. pombe (with S. Hauf, FMI/MPI)

Major mitotic kinases

Adapted from: Alexander et al. 2011. Science Signaling Vol 4 Issue 179 ra42

Aurora kinase family

Mammals A. thaliana X. laevis D. melanog. C. elegans S. cerevisiae S. pombe

Aurora A

AtAur1

AtAur2

AtAur3

EG2 Aurora AIR-1

Aurora B AIRK2 IAL AIR-2 Ipl1 Ark1

Aurora C

Yeast Aurora or metazoan Aurora B functions and substrates

AuroraAurora

PP

P

P

P

P

P

P

Condensin Condensin

Known substrates: Histone H3 (Ser10)Known substrates: Histone H3 (Ser10)

Other substrates? Other substrates? Spindle assembly checkpointSpindle assembly checkpoint

P

PP

P PRegulation of kinetochore-microtubule attachment

Regulation of kinetochore-microtubule attachment

Outer kinetochore proteins: Ndc80/Hec1, Dsn1, KNL1 Dam1/DASH complex MCAK

Outer kinetochore proteins: Ndc80/Hec1, Dsn1, KNL1 Dam1/DASH complex MCAK

Compaction of chromosomesCompaction of chromosomes

G2

M

G2 arrest (cdc25ts)

Exp. 1: Aurora inhibition

Aurora-as

+ microtubule drug

Exp. 2: Aurora inhibition+ microtubule drug

Aurora-as

Exp. 3: Inhibitor side effects

wild type Aurora

Replicates and Control

0-2-4-6 2 4 6

Exp. 1 + 2: Aurora inhibition

Exp. 3: Inhibitor side effects

log2 (H/L)

quantified: 5428

log 1

0 (in

tens

ity)

log2 (H/L)

quantified: 4877

log 1

0 (in

tens

ity) ✔ ✖

0-2-4-6 2 4 6

Quantitation Results: Phosphoproteome

Quantitation Results: Proteome

Classification of downregulated P-sites

Class Exp1 Exp2 Exp3 counts P-sites proteins

Class 1 -1 -1 0 45 45 24

Class 2 - -1

-1-

00

1015 25 23

Class 3

-1 -1 0 1

01-1-1

0000

421

451

89 65

Class 4

-1 -

-1 -1 -1 0

-1-1-01-1

------

19

161123

42 33

-1 = downregulated0 = 1:11 = upregulated- = not detected

Known substrates identified

AuroraAurora

PP

P

P

P

P

P

P

Condensin Condensin

Known substrates: Histone H3Known substrates: Histone H3

P

PP

P P

Outer kinetochore proteins: Ndc80/Hec1, Dsn1, KNL1 Dam1/DASH complex MCAK

Outer kinetochore proteins: Ndc80/Hec1, Dsn1, KNL1 Dam1/DASH complex MCAK

Known substrates: Histone H3Known substrates: Histone H3

Condensin Condensin

Outer kinetochore proteins: Ndc80/Hec1, Dsn1, KNL1 Dam1/DASH complex MCAK

Outer kinetochore proteins: Ndc80/Hec1, Dsn1, KNL1 Dam1/DASH complex MCAK

✔

✔

✔

Refinement of Aurora kinase target sequence

Currently accepted target sequences:

[RK]-X-[ST]-[ILV] (S. serevisiae)[RKN]-R-X-[ST]-[ILVM] (human Aurora-A)

R-X-[ST] R-K-R-X-[ST] R-X-[ST]

Novel Aurora kinase substrates in S. pombe

AuroraAurora

PP

P

P

P

P

P

P

P

PP

P PRegulation of kinetochore-microtubule attachment

Regulation of kinetochore-microtubule attachment

Compaction of chromosomesCompaction of chromosomes

P

P

Modulation of DNA damage response

Modulation of DNA damage response

‘Clearing’ of chromatin, facilitating segregation‘Clearing’ of chromatin, facilitating segregation

Inheritance of heterochromatin, preserving differentiated state

Inheritance of heterochromatin, preserving differentiated state

Setting DNA replication pattern

Setting DNA replication pattern

P

Spindle assembly checkpointSpindle assembly checkpoint

Novel functions of Aurora kinase

Example 2: Use of dominant-negative kinase mutants

Detection of PKD targets in HEK 293 cells (with A. Hausser, Uni Stuttgart)

Detection of PKD1 substrate candidates

From: Fu and Rubin. 2011. EMBO Reports 12(8): 785-796.

PKD1: cytosolic serine/threonine-protein kinase • converts DAG signals into prolonged physiological effects downstream of PKC• regulation of MAPK8/JNK1 and Ras signaling • Golgi membrane integrity and trafficking• cell survival through NF-kappa-B activation • cell differentiation by mediating HDAC7 nuclear export • cell proliferation via MAPK1/3 (ERK1/2) signaling

Detection of PKD substrate candidates

PKD motif among significantly regulated sites

PKD-dependent phosphorylation events

Reproducibility

[PKDca/PKDkd]Noco+ [parental/PKDkd]Noco+

Kinase of interest must be active during experiment

[PKDca/PKDkd]Noco+ [parental/PKDkd]Noco+

Importance of normalization by protein ratio

Overexpressed PKDkd

Enrichment of PKD target sequence

term p p.adj direction m x N k Category proteinsmembrane organization 0,000908619 0,039459278 enrichment 64 7 5851 144 GOBP.NamesIPI00021405;IPI00024417;IPI00029601;IPI00063784;IPI00220527;IPI00289819;IPI00299095Golgi apparatus 0,00115072 0,039459278 enrichment 151 11 5851 144 GOCC.NamesIPI00006211;IPI00014219;IPI00016780;IPI00032971;IPI00063784;IPI00165651;IPI00220527;IPI00290337;IPI00301280;IPI00303882;IPI00472533integral to membrane 0,001261707 0,039459278 enrichment 725 31 5851 144 GOCC.NamesIPI00006211;IPI00009235;IPI00746666;IPI00014218;IPI00018071;IPI00022558;IPI00029002;IPI00030634;IPI00034277;IPI00063784;IPI00152700;IPI00165651;IPI00169267;IPI00218922;IPI00220835;IPI00289819;IPI00292135;IPI00294501;IPI00296938;IPI00301280;IPI00328715;IPI00374657;IPI00385267;IPI00395887;IPI00419221;IPI00455473;IPI00604430;IPI00640341;IPI00746934;IPI00782950;IPI00792065nuclear membrane 0,001448047 0,039459278 enrichment 51 6 5851 144 GOCC.NamesIPI00032358;IPI00294501;IPI00301280;IPI00917683;IPI00328715;IPI00792065steroid metabolic process 0,002471298 0,053874297 enrichment 39 5 5851 144 GOBP.NamesIPI00014219;IPI00024971;IPI00032971;IPI00163644;IPI00294501tubulin binding 0,003442023 0,062530078 enrichment 42 5 5851 144 GOMF.NamesIPI00003420;IPI00006211;IPI00027963;IPI00152946;IPI00796333PH domain 0,005346906 0,083258961 enrichment 87 7 5851 144 Pfam.DescriptionsIPI00024971;IPI00029834;IPI00032971;IPI00065931;IPI00163644;IPI00939694;IPI00782950membrane invagination 0,00732811 0,094221322 enrichment 50 5 5851 144 GOBP.NamesIPI00024417;IPI00029601;IPI00220527;IPI00289819;IPI00299095intrinsic to membrane 0,007833636 0,094221322 enrichment 715 28 5851 144 GOCC.NamesIPI00006211;IPI00009235;IPI00746666;IPI00014218;IPI00018071;IPI00022558;IPI00029002;IPI00030634;IPI00034277;IPI00063784;IPI00152700;IPI00165651;IPI00169267;IPI00218922;IPI00220835;IPI00292135;IPI00294501;IPI00296938;IPI00301280;IPI00328715;IPI00385267;IPI00395887;IPI00419221;IPI00455473;IPI00604430;IPI00746934;IPI00782950;IPI00792065cell proliferation 0,008644158 0,094221322 enrichment 52 5 5851 144 GOBP.NamesIPI00003420;IPI00015104;IPI00063784;IPI00290435;IPI00782950

Enrichment of GO terms

Refinement of PKD target sequence

Currently accepted target sequence:[LVI]-X-[RK]-X-X-[ST]

[LV]-K-K-K-L-[ST] X-X-K-X-X-[ST]Depletion of Pro!

Acknowledgements

Proteome Center TuebingenKarsten KrugMirita Franz-WachtelSilke Wahl

University of StuttgartAngelika HausserStephan Eisler

Friedrich Miescher LaboratorySilke HaufAndre Koch

Funding

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