MaxQuant Summer School
Martinsried, June 26, 2013
Boris Maček
Proteome Center Tübingen
1

Central „dogma“ of molecular biology
Genome Transcriptome Proteome
Phosphorylation
Glycosylation
Disulfide bonds
Proteolysis
Acetylation
Methylation
Sulfation
Ubiquitination
GPI Anchor etc...
2

Most (if not all) proteins are modified
Regulatory modifications are dynamic:
• kinase/phosphatase
• acetyltransferase/deacetylase
• ubiquitin ligase/deubiquinating enzyme
• glyosyltransferase/deglycosidase
3
Figure 3-64 Molecular Biology of the Cell (© Garland Science 2008)

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

PTMs analyzed in large scale by MS
Choudhary and Mann. 2010. Nature Rev Mol Cell Biol. 11:427
5

Largest phosphoproteomes reported so far
Choudhary and Mann. 2010. Nature Rev Mol Cell Biol. 11:427
6

Gel-free phosphoproteomics workflow
Antibodies:
4G10 pY20, pY99, pY100
7

1st stage of enrichment:
Strong Cation Exchange (SCX) chromatography
Beausoleil et al. 2004. PNAS 101(33): 12130-12135
8

1st stage of enrichment:
Strong Cation Exchange (SCX) chromatography
9

2nd stage of enrichment:
IMAC or TiO
2 chromatography
10

Quantification of PTMs
Unmodified peptide
(change in protein level)
Modified peptide
(change in modification level)
No change
Upregulation
Downregulation
11

Quantification of PTMs
Unmodified peptide
(change in protein level)
Modified peptide
(change in modification level)
No change
Downregulation
(position?)
12

Intracellular signaling networks:
EGFR Signaling Pathway
13

Global phosphorylation dynamics
Main conclusions:
The EGF induced phosphorylation signal spreads to many different protein classes within 20 min of stimulation
6600 phosphorylation-sites from more than 2000 proteins pS (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
14
Olsen et al., Cell 2006, Volume 127, Issue 3 , p. 635-648

Detection of kinase substrate candidates
• 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 substrates:
Kinase inactivation
Control cells
WB
Kinase Inactivation
WB
Adapted from Kettenbach et al. Science Signaling 2011, Vol 4 Issue 179 rs5
Kinase inactivation:
1) By chemical inhibition
2) By overexpession of inactive kinase („dominant-negative“ mutant)
3) By gene modification (analog-sensitive kinases)
4) By gene knockout ( only non-essential kinases!
)

Detection of kinase substrates
”normal AA”
Lys12 C
6
14 N
2
”heavy AA” (+8Da)
Lys13 C
6
15 N
2
Kinase Inactivation
Control cells
Combine and lyse
GeLC-MS
(15 slices)
Proteolysis
(Lys-C)
SCX
TiO
2 nanoLC-MS/MS

Dominant–negative kinase mutants
• overexpression of inactive kinase → suppression of endogenous kinase activity
(also called „kinase-dead“ strains)
• problem: some endogenous kinase activity remains
Example: Receptor Tyrosine Kinases
Figure 15-53, 15-54 Molecular Biology of the Cell (© Garland Science 2008)

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
Aurora A
A. thaliana
Aurora B
AtAur1
AtAur2
AtAur3
Aurora C
X. laevis
EG2
D. melanog.
Aurora
C. elegans
AIR-1
S. cerevisiae S. pombe
AIRK2 IAL AIR-2 Ipl1 Ark1

Yeast Aurora or metazoan Aurora B functions and substrates
Regulation of kinetochoremicrotubule attachment
Compaction of chromosomes
Spindle assembly checkpoint
P
P
P
P
P
P
P
P
P
P
P
P
P
Known substrates:
 Histone H3 (Ser 10 )
 Outer kinetochore proteins:
Ndc80/Hec1, Dsn1, KNL1
Dam1/DASH complex
MCAK
 Condensin
 Other substrates?

G2
Replicates and Control
Exp. 1: Aurora inhibition Exp. 2: Aurora inhibition
+ microtubule drug
Exp. 3: Inhibitor side effects
G2 arrest ( cdc25ts )
Aurora-as Aurora-as wild type Aurora
M
+ microtubule drug

Quantitation Results: Phosphoproteome
Exp. 1 + 2:
Aurora inhibition quantified: 4877
✔
Exp. 3:
Inhibitor side effects quantified: 5428
✖
-6 -4 -2 0 log
2
(H/L)
2 4 6 -6 -4 -2 0 log
2
(H/L)
2 4 6

Quantitation Results: Proteome

Classification of downregulated P-sites
-
-1
0
1
Class
Class 1
Class 2
Class 3
Class 4
Exp1
-1
-
-1
-1
-1
0
1
-1
-
-1
-1
-1
0
= downregulated
= 1:1
= upregulated
= not detected
Exp2
-1
-1
-
0
1
-1
-1
-1
-1
-
0
1
-1
Exp3
0
0
0
0
0
0
0
-
-
-
-
-
counts
45
10
15
42
1
45
1
1
9
16
11
2
3
P-sites proteins
45 24
25 23
89
42
65
33

Known substrates identified
P
P
P
P
P
P
P
P
P
P
P
P
P
 Histone H3
✔
 Outer kinetochore proteins:
✔
 Condensin
✔

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

Novel functions of Aurora kinase
P
Regulation of kinetochoremicrotubule attachment
Compaction of chromosomes
Spindle assembly checkpoint
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
‘Clearing’ of chromatin, facilitating segregation
Modulation of
DNA damage response
Inheritance of heterochromatin, preserving differentiated state
Setting DNA replication pattern

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

Detection of PKD substrate candidates

PKD-dependent phosphorylation events
PKD motif among significantly regulated sites

Reproducibility

Kinase of interest must be active during experiment
[PKDca/PKDkd] Noco+ [parental/PKDkd] Noco+

Importance of normalization by protein ratio
[PKDca/PKDkd] Noco+ [parental/PKDkd] Noco+
Overexpressed PKDkd

Enrichment of PKD target sequence term membrane organization
Golgi apparatus integral to membrane nuclear membrane steroid metabolic process tubulin binding
PH domain membrane invagination intrinsic to membrane cell proliferation p
Enrichment of GO terms
0,000908619
0,00115072
0,001261707
0,001448047
0,002471298
0,003442023
0,005346906
0,00732811
0,007833636
0,008644158 p.adj
direction m
0,039459278 enrichment
0,039459278 enrichment
0,039459278 enrichment
0,039459278 enrichment
0,053874297 enrichment
0,062530078 enrichment
0,083258961 enrichment
0,094221322 enrichment
0,094221322 enrichment
0,094221322 enrichment
39
42
87
50
64
151
725
51
715
52 x
28
5
5
5
7
5
7
11
31
6
N
5851
5851
5851
5851
5851
5851
5851
5851
5851
5851 k Category proteins

Refinement of PKD target sequence
Currently accepted target sequence:
[LVI]-X-[RK]-X-X-[ST]
[LV]-K-K-K-L-[ST]
Depletion of Pro!
X-X-K-X-X-[ST]

Acknowledgements
Proteome Center Tuebingen
Karsten Krug
Mirita Franz-Wachtel
Silke Wahl
University of Stuttgart
Angelika Hausser
Stephan Eisler
Friedrich Miescher Laboratory
Silke Hauf
Andre Koch
Funding
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