Enrichment and Six-Plex Profiling of the DNA Damage Response

Enrichment and Six-Plex
Six Plex Profiling
g of the DNA Damage
g Response
p
Pathway
y Using
g AmineAmine and
Cy t i R
Cysteine-Reactive
ti T
Tandem
d
M
Mass Tag
T g Reagents
R g t
Ryan
y D
D. Bomgarden
Bomgarden,
g
Michael M.
M Rosenblatt,
Rosenblatt John C.
C Rogers
g
Th
Thermo
Fisher
Fi h Scientific,
S i tifi Rockford,
R kf d IL,
IL USA
Overview
Purpose: To identify and characterize the DNA damage
damage-response
response proteins
in pulmonary carcinoma A549 cells in response to five different anti
anti-cancer
cancer
agents
agents.
Methods: Six-plex, amine-reactive Tandem Mass Tag (TMT) reagents
were used for total proteome
p
labeling
g and relative quantitation
q
among
g
ttreatments.
eat e ts S
Six-plex
p e cyste
cysteine-reactive
e eact e Tandem
a de Mass
ass Tag
ag (cys
(cysTMT))
reagents were used for subproteome peptide labeling followed by
enrichment using a novel immobilized anti
anti-TMT
TMT antibody resin
resin.
Results: Using both amine- and cysteine-reactive TMT reagents, we
identified over 4700 peptides
p p
(FDR
(
< 1%)) and q
quantitated the relative
abundance
abu
da ce o
of o
over
e 1000
000 proteins
p o e s of
o A549
5 9 cells
ce s treated
ea ed with DNA-damaging
da ag g
agents. However, only changes in the most abundant DNA damage
damageregulated proteins were observed in un-enriched
un enriched samples labeled with
TMT CysTMT reagent
TMT.
reagent-enriched
enriched samples resulted in the identification and
quantitation of additional proteins whose levels were altered after drug
t t
treatment.
t
I t d ti
Introduction
The DNA damage-response pathway is critical in maintaining genome
stability, and proteins within this pathway are commonly mutated or misregulated
g
in cancer cells (Figure
( g
1).
) 1 We used a mass spectrometry
p
y ((MS))
based p
proteomic
oteo c approach
app oac to identify
de t y and
a d characterize
c a acte e the
t e DNA da
damageage
response proteins in pulmonary carcinoma A549 cells in response to five
anti-cancer
anti
cancer agents: camptothecin,
camptothecin etoposide,
etoposide actinomycin D
D, hydroxyurea
hydroxyurea,
and paclitaxel (Figure 2)
2). Each of these drugs has been used clinically to
treat cancer and has distinct mechanisms of action.
action Camptothecin and
etopiside
t i id are ttopoisomerase
i
II and
d I iinhibitors,
hibit
respectively,
ti l and
d preventt
unwinding
i di off DNA d
during
i replication
li ti in
i S
S-phase.
h
A
Actinomycin
ti
i D,
D an RNA
polymerase II inhibitor, and hydroxyurea, a dNTP synthesis inhibitor, are
also S-phase inhibitors. Paclitaxel is a microtubule stabilizer that arrests
cells in mitosis. We used TMT reagents
g
to determine relative changes
g in
overall
o
e a protein
p ote abundance
abu da ce after
a te each
eac drug
d ug ttreatment.
eat e t
Tandem
T
d
M
Mass Tag
T reagents
t enable
bl concurrentt id
identification
tifi ti and
d
multiplexed quantitation of proteins in different samples using tandem MS.2
Isobaric TMT reagents share identical structures, including a peptidereactive labeling
g group,
g p, a spacer
p
arm and an MS/MS reporter.
p
During
g
MS/MS
S/ S analysis,
a a ys s, each
eac isobaric
soba c tag
ag produces
p oduces a unique
u que reporter
epo e ion
o that
a
enables quantitation. Current amine
amine-reactive
reactive NHS
NHS-ester
ester duplex or six-plex
six plex
isobaric TMT reagents covalently attach to the free amino termini and
lysine residues of peptides and proteins
proteins. Our cysTMT reagents and an
anti TMT antibody resin provide an approach of thiol labeling,
anti-TMT
labeling affinity
enrichment,
i h
t and
d quantitation
tit ti similar
i il to
t isotope-coded
i t
d d affinity
ffi it tags
t
(ICAT™) 3 Overall,
(ICAT™).
O
ll targeted
t
t d cysteine
t i labeling
l b li and
d affinity
ffi it enrichment
i h
t using
i
cysTMT reagents results in reduced sample complexity and improved
proteome depth compared to the amine-labeled proteome.
Methods
Sample Preparation
Cell treatment and Western blot: A549 cells grown in DMEM with 10%
FBS were treated with 10 μM
μ camptothecin,
p
, 200 μM
μ etoposide,
p
, 2 μM
μ
actinomycin
act
o yc D,, 10
0 mM hydroxyurea,
yd o yu ea, o
or 10
0 μg/
μg/ml paclitaxel
pac ta e for
o 20
0 hours
ou s at
37 ºC
C. A549 cell lysates (10 μg) were separated by SDS-PAGE
SDS PAGE and blotted
using Thermo Scientific p53 antibodies diluted 1:10,000.
1:10 000
TMT® reagent
g
labeling:
g Control or treated A549 cells were sonicated in
lysis
ys s buffer
bu e (6 M urea/
u ea/ 50 mM Tris/
s/ 5 mM EDTA)) and
a d reduced
educed with
t 5 mM
DTT for 1 hour at 50 ºC and alkylated with 375 mM iodoacetamide for
15 minutes at room temperature.
temperature Each sample was acetone precipitated and
enzymatically digested at 37 ºC
C overnight
overnight. Peptides were labeled with the
appropriate TMTzero® or TMTsixplex® reagent (126
(126-131)
131) for 2 hours at
room temperature,
t
t
quenched
h d with
ith hydroxylamine,
h d
l i
and
d combined
bi d before
b f
li id chromatography
liquid
h
t
h (LC)-MS/MS
(LC) MS/MS analysis.
l i
cysTMT® reagent labeling and enrichment: Treated A549 cells were
lysed and reduced as above.
above Reductant was removed using Thermo
Scientific Zeba 7K Desalting Columns
Columns, and 100 μg of each sample was
immediatel combined with
immediately
ith 20 μL
L of 25 mM ccysTMT
sTMT reagent
reagent. Each sample
was labeled
l b l d with
ith the
th appropriate
i t cysTMTzero®
TMT
® or cysTMTsixplex®
TMT i l ® reagentt
(126-131) for 2 hours at room temperature, combined, and separated by
6%-12% SDS-PAGE. Eight gel bands were separately enzymatically digested
at 37 ºC overnight
g before extraction and lyophylization.
y p y
Labeled peptides
p p
were resuspended
p
in TBS and incubated with 50 μL
μ of immobilized anti-TMT
antibody resin per gel band overnight with end
end-over-end
over end shaking at 4 ºC.
C.
After collecting the unbound sample
sample, the resin was washed four times with
4 M urea/TBS
urea/TBS, four times with 0
0.05%
05% CHAPS
CHAPS, four times with TBS
TBS, and four
times with water
water. Peptides were eluted four times with 50% acetonitrile
acetonitrile,
0 4% TFA and vacuum-dried
0.4%
ac m dried before LC
LC-MS/MS
MS/MS anal
analysis.
sis
LC/MS
A NanoLC-2D™ high-pressure liquid chromatograph (HPLC) (Eksigent) with
a Proteopep™II C18 column, 75 µm ID x 20 cm (New Objective) was used
to separate
p
peptides
p p
using
g a 4%-40% g
gradient ((A: water,, 0.1% formic acid;;
B: acetonitrile,, 0.1%
% formic acid)) at a flow rate of 250 nL/min for 60 min. A
Thermo Scientific LTQ Orbitrap XL ETD mass spectrometer was used to
detect peptides using a top 2 x 3 experiment consisting of single-stage
single stage MS,
MS
followed by acquisition of three MS/MS spectra with higher-energy C-trap
dissociation (HCD) fragmentation,
fragmentation followed by three MS/MS with collision
ind ced dissociation (CID) for protein identification
induced
identification. Parameters in this mode
were: Isolation
I l ti width:
idth 3.0
3 0 m/z;
/ C
Collision
lli i energy: 50% (10% with
ith two
t
steps).
t
)
Only doubly- and triply-charged peptides were selected for fragmentation.
Dynamic exclusion parameters were set to: Repeat count = 1; Repeat
duration = 30;; Exclusion list size = 500;; Exclusion duration = 20. Target
g
values were as follows: MS = 5 e5;; MS/MS (HCD)
(
) = 1 e5. Ion transfer times
were set to 500 for FTMS and 300 for MS/MS (HCD). Two microscans were
required for HCD spectra
spectra.
To determine relative changes
g in abundance of other p
proteins affected by
y
drug treatment, we used both amine
amine- and cysteine-reactive
cysteine reactive TMT reagents
(Figure 3).
3) Six-plex
Six plex amine-reactive
amine reactive TMT reagents were used for total
proteome labeling of peptides derived from each treatment
treatment. A set of six
sixplex cysTMT reagents were used to label the cysteine
cysteine-containing
containing
s bproteome In contrast to amine
subproteome.
amine-reactive
reacti e TMT reagents
reagents, these tags
were used
d to
t label
l b l denatured
d
t d proteins
t i before
b f
sample
l mixing
i i and
d
enzymatic digestion. Because of the relatively low abundance of
cysteine-containing peptides, approximately 6% of peptides are labeled,
allowing
g for potential
p
sampling
p g of lower abundant proteins.
p
Enrichment of
cysTMT
y
reagent-labeled
g
peptides
p p
was facilitated byy an immobilized antiTMT antibody resin, which has affinity for the TMT reagent reporter
region (Figure 3)
3).
Using these parallel workflows, we were able to identify over 4700
peptides
p
p
and q
quantifyy over 1000 proteins
p
(Figure
( g
2C and 4).
) Onlyy 541
proteins were similar between the amine- and cysteine-labeled
p
y
samples,
p ,
demonstrating increased sample diversity after enrichment. Unique to the
cysTMT enriched sample,
sample were some known and novel DNA damagedamage
response proteins,
proteins which resulted in changes in relative abundance after
drug treatment
treatment. Specifically,
Specifically two DNA damage signaling targets
targets, ASC
ASC-1
1
and aslin 1
1, were
ere red
reduced
ced o
over
er ttwo-fold
o fold in response to camptothecin
camptothecin,
etoposide
t
id and
d actinomycin
ti
i D (Figure
(Fi
5A,
5A 5B).
5B) A similar
i il reduction
d ti
occurred for the DNA helicase MCM complex, with subunits 2-6 being
quantified after enrichment (Figure 5C and data not shown). Some
proteins such as the HUWE1,, a p
p
p53 E3 ubiquitin
q
ligase
g
like MDM2,, had
increased expression
p
upon
p drug
g treatment (Figure
( g
5D).
) Others such as
the small GTPase guanidine exchange factor RalBP1, had drug-specific
drug specific
down regulation (Figure 5E).
5E)
FIGURE 5. Identification and quantitation
q
of Thermo Scientific
cysTMT
y
reagent-labeled
g
peptides.
p p
A
ASC-1 complex subunit P100
HDFcYR
B
Aslin 1
sFcTGKSDSR
F TGKSDSR
Data analysis: Mixed CID and HCD data were processed with Thermo
Scientific Proteome Discoverer™ 1.2 software against
g
the IPI human
database using
g a 20 pp
ppm mass tolerance. Static modifications were set to
the TMTsixplex (229.16 Da) or cysTMTsixplex reagent (304.18 Da) and
dynamic modifications included phosphorylation and methionine oxidation.
oxidation
C
R
Results
lt
D
MCM3
YVLcTAPR
E3 ubiquitin
biq itin ligase HUWE1
KVIcTGPNDtSPGsPR
Multiple genomic and proteomic studies have identified
f
roles for
f p53 in
regulation of cell cycle arrest, DNA repair, and apoptosis. Activation of the
p53 DNA damage-response
p
g
p
pathway
p
y causes p53
p stabilization through
g
phosphorylation,
p
p y
, leading
g to transcription
p
of various p53-regulated
p
g
p
proteins
(Figure 1). Treatment of A549 cells with five anti
anti-cancer
cancer agents,
camptothecin etoposide
camptothecin,
etoposide, actinomycin D
D, hydroxyurea
hydroxyurea, and paclitaxel
resulted in varying degrees of p53 activation (Figure 2B)
2B), which is consistent
with their potency and mechanism of action.
action
E
RalBP1
MELcGATRLGyFGR
(A-E) MS spectra of enriched cysTMT-labeled cysteine-containing peptides and TMT reporter
quantitation showing changes in relative abundance of peptides after each drug treatment.
FIGURE 3. Schematic of TMT and cysTMT reagent workflows.
A
C
Conclusions
l i
FIGURE 1.
1 Model
M d l off p53
53 DNA damage
d
response pathway.
th
•
AmineA
i
and
d cysteine-reactive
t i
ti T
Tandem
d
M
Mass T
Tags are
complementary reagents that ffacilitate more in-depth proteomic
analysis.
Mdm2
•
cysTMT reagents combined with immobilized anti-TMT
anti TMT resin
enrichment allow for relative quantitation of lower abundant
proteins.
t i
Apoptosis
•
Six plex TMT reagents enabled simultaneous relative quantitation
Six-plex
of five different anti-cancer drugs
drugs.
•
Combined CID and HCD spectra
p
processing
p
g facilitated enhanced
quantitative peptide
q
p p
identification.
DNA damaging agents (Campto/Etop/ActD)
p
B
p53
p53 protein levels
Cell survival
Cell cycle arrest & genetic repair
Activation of the p53 DNA damage-response
g
pathwayy results in p53 phosphorylation
y
and stabilization
through interference with MDM2-mediated degradation. Increased p53 levels result in increased expression
of stress response genes that promote cell survival through cell cycle arrest or DNA damage repair. p53
activation also induces expression of pro-apoptotic genes and the MDM2 ubiquitin E3 ligase which in turn
modulates the down-regulation of the pathway.
pathway
(A) Si
Six-plex
l TMT reagentt workflow
kfl
ffor peptide
tid llabeling
b li after
ft sample
l treatment
t t
t and
d digestion.
di
ti
(B) SixSi
plex cysTMT reagent workflow for protein labeling followed by digestion and labeled peptide enrichment
using immobilized anti-TMT antibody resin.
FIGURE 2. A549 cell drug treatments and response.
A
FIGURE 4. Comparison of TMT and cysTMT reagent
reagent-enriched
enriched
peptides and proteins.
C
Cell
C
ll cycle
l
phase TMT reporter
[]
No treatment
-
-
126
Camptothecin
10 μM
S
127
Etoposide
Actinomycin D
Hydroxyurea
Paclitaxel
200 μM
S
128
2 μM
S
129
10 mM
S
130
10 μg/ml
M
131
Fold change
c
e
Drug
10
127/126
128/126
129/126
130/126
131/126
B
TMT Total cysTMT Enriched
3604 Peptides
p
1
0
100
200
300
935 Peptides
p
TMT Total
cysTMT Enriched
823 Proteins
781 Proteins
400
0.1
Protein Number
B
A
3522
82
1152
282
541
240
p53
p
-
Capto Etop ActD
HU
Pax
(A) Table of different anti-cancer agents and concentrations used for A549 cells treatment, the cell cycle
phase of drug action,
action and the TMTsixplex or cysTMTsixplex reagents used to label each sample
sample. (B)
Western blot of p53 after drug treatment. (C) Log graph of relative quantitation of 400 cysTMT reagentlabeled proteins normalized to no treatment control
control.
4756 Total Peptides
1063 Total Proteins
Venn diagrams
g
depicting
p
g the number of unique
q labeled peptides
p p
(A)
( ) and proteins
p
(B)
( ) identified using
g
Scaffold 3 Q+ (Proteome Software) for TMT reagent-labeled samples and cysTMT reagent sample post
enrichment of A549 cell extract using immobilized anti-TMT
anti TMT resin.
References
1 Vousden
1.
Vousden, K
K.H.
H and Prives C
C. (2009)
(2009). Blinded by the light: The
growing complexity of p53
p53. Cell 137(3):413-431.
137(3):413-431
2. Thompson,
p
A., et al. ((2003).
) Tandem mass tags:
g a novel
quantification strategy
q
gy for comparative
p
analysis
y of complex
p
protein
p
mixtures by MS/MS. Anal. Chem. 75: 1895-204.
1895 204.
3 G
3.
Gygi,
i S.P.,
S P ett al.
l (1999).
(1999) Q
Quantitative
tit ti analysis
l i off complex
l protein
t i
mixtures using isotope-coded affinity tags. Nat. Biotech. 17: 994999.
Acknowledgements
The authors would like to thank Eugene Cichon for help with additional
sample preparation.
preparation
Tandem Mass Tag,
g, TMT,, TMTzero,, TMTsixplex,
p , cysTMT,
y
, cysTMTzero,
y
, and cysTMTsixplex
y
p
are registered
g
trademarks of Proteome
Sciences plc. ICAT is a trademark of the University of Washington. Eksigent is a registered trademark and NanoLC is a trademark of AB
SCIEX LLC.
LLC Proteopep is a trademark of New Objective
Objective. All other trademarks are the property of Thermo Fisher Scientific Inc
Inc. and its
subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual
property
t rights
i ht off others.
th