Protein Phosphorylation
and
Sample Prep
Johan Öhman
Senior Scientist
GE Healthcare Bio-Sciences AB
Outline
Introduction
Case Study 1 pervanadate treated CHO cells
Case Study 2 phosphoproteomics
Case Study 3 drug treatment of cancer cells
Summary
2
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
3
Background protein phosphorylation
~15% of total proteins
(some cellular state)
O
O
Differences between
Ser/Thr and Tyr
phosphorylations.
P
Ser Thr Tyr
1000 / 100 / 1
O
O
N
O
O O
P
O
O
N
O
Relative abundance:
OO
P
O
O
Tyrosinephosphate
N
O
Threonine-/
Serine-phosphate
4
Phosphorylation - research areas
Cellular processes
Therapeutic
• apoptosis
• cancer
• cell cycle / DNA repair
• diabetes / obesity
• kinases
• immunology
• signaling
• angiogenes
• oncogenes / tumor suppressors
• pathogenes
Other
• phospho proteomics
• interaction proteome
• localization and dynamics
5
Phosphorylation - detection
Techniques
•
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
6
Sample preparation
Workflow
Sample
specific
Downstream
Core
specific
procedure
Downstream application
Sample
Sample preparation
Analysis
7
Sample prep - improves the quality
Minimize
• degradation
• sample loss
optimize the
quality of
the sample
• contamination
• in vitro chemical modifications
… leads to enhanced
• signal for low abundance molecules
• analytical resolution
maximize the
quality of
the result
8
Sample prep - enrichment techniques
Sample mixture
Immunoprecipitation
pTyrantibody
pSer/pThrantibody
Affinity
matrix
IMAC
Chemical
modification
MOAC
Fe3+
Ga3+
Zr4+
Al3+
TiO2
9
Sample prep – workflow phosphoproteins
10
Sample prep - phosphoproteins
Important considerations
•
Phosphatase inhibitors
• Reduce phosphatase activity
•
Low abundance
Amount material needed:
•
Obscured (hidden) PO4 groups
100-200 fmol peptide in LC-MS/MS
•
Mapping protein interactions
10-200 million cells, 1-20 ml cell extract
•
Multi-protease approach may
be needed (LC-MS/MS)
•
Reduce ”background” by using
good controls
11
Mag
TM
Sepharose
Attractive sample preparation
made easy
12
Products for enrichment of proteins and
phosphopeptides
Magnetic beads
Additional material
Specific Ligands, e.g.:
Antibodies
Other Binders
NHS Mag Sepharose™
Protein A Mag Sepharose
Protein G Mag Sepharose
TiO2 Mag Sepharose
MagRack 6
Low µg scale
Variable amount of beads
Variable sample volume
Enzyme inhibitors, e.g.:
Protease inhibitors
Phosphatase inhibitors
Extraction and
immunoprecipitation buffers
Your Sample – Cells or tissue
13
Enrichment of
phosphoproteins and
peptides
Case study 1:
Finding low abundant
tyrosine phosphoproteins in
CHO cells
14
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
15
Sample prep - enrichment techniques
Sample mixture
Immunoprecipitation
pTyrantibody
pSer/pThrantibody
Affinity
matrix
IMAC
Chemical
modification
MOAC
Fe3+
Ga3+
Zr4+
Al3+
TiO2
16
Sample prep – workflow phosphoproteins
17
Changes in tyrosine phoshorylation
in CHO cells upon pervanadate treatment
Biological system
CHO cells
Treatment with pervanadate (H2O2 + vanadate), known to
introduce a decrease in tyrosine phosphorylation
Tools and methods
Phospo tyrosine specific antibody, PY20
Protein G Mag Sepharose™ beads
Trypsin
Mass spectrometry, LC-MS/MS
18
Enrichment of tyrosine phosphorylated
proteins in CHO cells
™
Trypsin digestion
19
Changes in tyrosine phoshorylation
in CHO cells upon pervanadate treatment
• 76 potential tyrosine
phosphoproteins identified
Single 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
Oxidative stress indicated !
20
Enrichment of
phosphopeptides
Case Study 2:
Phosphopeptides in a
leukemia cancer cell line
enriched by TiO2
21
Sample prep – workflow phosphoproteins
22
Enrichment and identification of
phosphopeptides in cancer cells
Workflow for analysis of phosphopeptides
from leukemia cell line
Cell lysis
Trypsin digestion of
proteins in cell lysate
Desalting of peptides
(C-18)
TiO2 Mag Sepharose ™
protocol
LC-MS/MS analysis and
peptide identification
23
Identified proteins & phosphopeptide sequences
Accession number
1
Gene name
Protein name
Phosphopeptide sequence
IPI00012442
G3BP1
Ras GTPase-activating protein-binding protein
1
IPI00009032
SSB
IPI00009032
2
3
Site
SwissProt
sssPAPADIAQTVQEDLR
S230/S231/S232
Yes, for all 3
Lupus La protein
FAsDDEHDEHDENGATGPVKR
S366
Yes, by CK2
SSB
Lupus La protein
TKFAsDDEHDEHDENGATGPVKR
S366
Yes, by CK2
IPI00025512
HSPB1
Heat-shock protein
QLsSGVSEIR
Nobeta-1
phosphopeptides identified
S82
Yes
IPI00185526
SAMSN1
SAM-domain protein SAMSN-1
before
S11
No
IPI00017297
MATR3
Matrin-3
RDsFDDRGPSLNPVLDYDHGSR
S188
Yes
IPI00184330
MCM2
DNA replication licensing factor MCM2
GLLyDSDEEDEERPAR
Y137 (or S139)
No, S139 known
IPI00017659
RCSD1
Enriched
Protein kinase substrate
CapZIP
S179
Yes, by MAPKAPK2
and MAPKAPK3.
IPI00023503
CDK3
Cell division protein kinase 3
Y15
Yes
IPI00013721
PRPF4B
binds
TiO2PRP4
Serine/threonine-protein kinase
homolog
Y849
Yes
IPI00337465
KLC1
Isoform P of Kinesin light chain 1
AssLNVLNVGGK
S546/S547
No, sequence
specific for isoform P
IPI00163505
RMB39
Isoform 1 of RNA-binding protein 39
DKsPVREPIDNLTPEER
S136
Yes
IPI00014177
Septin-2
Septin-2
IYHLPDAEsDEDEDFKEQTR
S218
Yes
IPI00299254
EIF5B
Eukaryotic translation initiation factor 5B
NKPGPNIEsGNEDDDASFK
S214
Yes
IPI00178667
TOP2A
183 kDa protein/DNA topoisomerase 2
yLEESDEDDLF
Y1601
No
SSsFGNFDR
enrichment
!
sample identified
SQsDCGELGDFR
15 phosphopeptides
IGEGTyGVVYK
to both
LCDFGSASHVADNDITPyLVSR
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.
24
Enrichment of
phosphoproteins and
peptides
Case Study 3:
Changes in tyrosine
phosphorylation in cancer cells
upon drug treatment
25
Sample prep – workflow phosphoproteins
26
Challenges for low abundant proteins
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
27
How can we improve the possibility for
detection of low abundant proteins?
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
28
Select a separation method for optimal
resolution of protein of interest
Method:
•1-D SDS-PAGE
•2-D electrophoresis
•1-D Western
•2-D Western
Parameters:
•pH interval
•Gel size
•Acryl amide content
•IEF gels
29
Quantification range
Deep Purple™
ECL Plex™
Sypro™ Ruby
Radio
isotopes
high
(pg)
CyDye™
Silver
sensitivity
Coomassie™
Blue
low
(ng)
30
What sets limit of detection?
Amount of sample
Signal intensity
Background
Variation in background (noise)
Signal intensity
(counts)
Signal: noise ratio ≥ 3 is limit of detection
Lane 22
Lane 23
Lane 24
Pixel position
Lane 25
31
Changes in tyrosine phoshorylation
in cancer cells upon drug treatment
Biological system
K562 chronic myeloid leukemia cells
Treatment with imatinib (Gleevec), known to introduce a decrease in tyrosine
phosphorylation
Questions
Can 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
32
Simplified DIGE experiment
CyDye™ pre-labeling
2-D Electrophoresis
Imaging
Total protein samples
Control (Cy™2)
Imatinib treated (Cy3)
33
No significant change in protein
abundances in total protein samples
control
treated
overlay
Immobiline™ drystrip: pH 4-7, 7 cm
SDS-PAGE gel: 4-20 % Tris Glycine, 8 x 7 cm
Data courtesy: Sara Lind, Rudbeck laboratory, Uppsala , Sweden
34
How can we improve the possibility for
detection of low abundant proteins?
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
35
Enrichment of proteins and peptides using
Mag Sepharose™ beads
NHS Mag Sepharose
Protein A Mag Sepharose
Protein G Mag Sepharose
TiO2 Mag Sepharose (peptides)
36
Analysis of low abundant phosphoproteins
Tools and methods
Phospho 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
37
Work flow enrichment of pTyr proteins
Protein G Mag Sepharose™
Anti phospho tyrosine antibody, 4G10
Overnight incubation with K562 cell lysate
at +4ºC
Phenyl phosphate (phospho tyrosine analogue)
Desalting, concentration and
exchange into DIGE labeling buffer
Vivaspin ultracentrifugation columns
(MWCO 5 kDa)
VivaSpin™
38
Down regulation detected by DIGE after
enrichment
control
treated
overlay
Protein G Mag Sepharose™ + anti pTyr 4G10
Immobiline™ drystrip: pH 4-7, 7 cm
SDS-PAGE gel: 4-20 % Tris Glycine, 8 x 7 cm
39
DIGE results before and after enrichment
Non-enriched
Immobiline™ drystrip: pH 4-7, 7 cm
SDS-PAGE gel: 4-20 % Tris Glycine, 8 x 7 cm
Enriched
Protein G Mag Sepharose™ + anti pTyr 4G10
Immobiline™ drystrip: pH 4-7, 7 cm
SDS-PAGE gel: 4-20 % Tris Glycine, 8 x 7 cm
40
Many tyrosine phosphoproteins are
down regulated upon drug treatment
Control
Imatinib treated
overlay
DeCyder™ 2-D differential analysis software
41
Large down regulation
Control
Imatinib
treated
Fold change
-5.14
-11.52
-11.13
42
Protein Identification results
Ratio
control/treated
Spot
no
Protein
Protein full name
Protein
accession1
Gel 12
Gel 22
1
-
n/a
-
-2.68
-2.07
2
-
n/a
-
-2.93
-1.94
3
CRKL
v-crk sarcoma virus CT10 oncogene
homolog (avian)-like
gi 4885153
-2.64
-2.47
n/a
-
-2.93
-1.95
4
5
TPM3
Tropomyosin 3 isoform 5
gi 114155148
-3.09
-1.48
6
14-3-3 ε
14-3-3 protein epsilon
gi 5803225
-5.14
-9.98
7
14-3-3 γ
14-3-3 protein gamma
P61981
-11.62
-11.56
8
-
n/a
-
-3.14
-8.81
9
-
n/a
-
-2.65
-2.39
GRB2
Growth factor receptor-bound protein 2
isoform 1
gi 4504111
-11.13
-2.33
10
43
2-D Western blotting showed decrease
in tyrosine phosphorylation
Total protein pre-labeled with Cy™3
Unlabeled total protein
Control
Cy 3
2-D electrophoresis
Transfer to membrane
Drug treated
Antibody probing
Cy 5
P
Anti phospho tyrosine primary, 4G10
ECL Plex™ Cy 5 secondary
Membrane image
Cy 3/Cy 5 overlay
44
Conclusions, case study 3
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
45
Summary
Enrichment of low abundant
phosphoproteins is necessary for
detection and identification
Good sample prep methods are
necessary for high quality
results
46
Thank You for your attention!
47
Questions!
48
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49
Recently Launched
Sample Prep Products
Target protein analysis in crude samples –
protein activity
Desalting
Concentration
Crude sample
Conditioning
Various assays
Know your protein activity
51
Antibody binding –Fluorescent Western
blotting
Quantification of target
protein relative to an
internal, endogenous
standard
ECL Plex™
RuvB
(house-keeping protein)
His- GFP
(target protein)
t0
t1
t2
t3
t4
Relative increase of His-GFP
Multiplexed fluorescent
detection for reliable
quantification
52
Antibody bindingFluorescent Western blotting
C
y™
5
y
C
5
C
y
3
y
C
3
ECL Plex™ secondary antibody
CyDye™ Conjugated
Primary antibody
Targets on membrane
= Target protein
= House-keeping protein
(internal standard)
53
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
54
Target protein analysis in purified sample
Target protein
property
Assay
Amino acid sequence
Chemical or MS/MS
Mass
MS
Protein activity
Depending on type of
activity
Western blotting, ELISA
etc.
UV/VIS spectroscopy
Antibody binding
Unique fluorescence /
absorbance
Size / apparent molecular SDS-PAGE
weight
Gel Filtration
Decreasing
specificity
55