Supplemental Information
Materials and Methods
p21 Pulse Chase Experiment
Control and C/EBPα knockdown keratinocytes were placed in Chelax-treated DMEM without Lmethionine and L-cysteine (Life Technologies) for 1 h, then pulsed labeled for 2 h by the
addition of 0.1 mCi/ml (0.4 mCi total) 35S-methionione –cysteine labeling mix (NEG772,
PerkinElmer). After labeling cells were incubated in normal growth media (t=0) and collected at
the indicated times after labeling. p21 was immunoprecipitated, subjected to SDS-PAGE,
incubated in Enlightening (GNE9741, GE Healthcare Life Sciences) and detected by
phosphorimaging with results presented as the percentage of
35
S-labelled p21 remaining
relative to the amount at t=0.
Chemicals
Formic acid, ammonium bicarbonate, DTT, hydrochloric acid, sodium deoxycholate and
iodoacetamide were obtained from Sigma Aldrich. Proteomics grade trypsin was purchased
from Promega. HPLC grade acetonitrile and water were from Burdick and Jackson.
LC MS/MS Sample Preparation
A modified filter aided sample preparation procedure was utilized.
First 80 µL of 1% sodium
deoxycholate in 50 mM ammonium bicarbonate (pH=8.0) was added to a 30 kDA MWCO filter
(Sartorius Corp). The Co-IP eluent was then added and mixed via several aspirations. Proteins
were reduced at 60ºC for 30 minutes upon addition of DTT to a final concentration of 5 mM.
One hundred microliters of 8M urea in 50 mM ammonium bicarbonate was added to the solution
and centrifuged at 14000g for 15 minutes. This process was repeated with the addition of 200
µL of 8M urea and proteins were alkylated in the dark for 20 minutes upon addition of
iodoacetamide to a final concentration of 15 mM. The sample was then washed with 100 µL of
8M urea followed by two additional washes with 200 µL of 50 mM ammonium bicarbonate.
Samples were digested with 200 ng of trypsin in 50 µL of 50 mM ammonium bicarbonate (pH=8)
for 4 hrs at 37ºC. Samples were collected after centrifugation (10 mins @ 14000 g) of the
digest and an additional 20 µL of wash. Samples were then acidified with the addition of 6 M
HCl to a final concentration of 200 mM (pH<3). Samples were injected (5µL), washed onto a 3
cm self packed trap, and separated with a 20 cm self-packed PicoFrit column (New Objective,
Woburn, MA) using an Easy nanoLC 1000 (Thermo Scientific, San Jose, CA). Both trap and
column were packed with 3 µm C18 reversed phase particles (Dr. Maisch GmbH, AmmerbuchEntringen, Germany). Mobile phase A and B were 98/2 Water/Acetonitrile 0.1% formic acid and
100% acetonitrile 0.1% formic acid, respectively. The flow rate was set to 300 nL/min. A linear
ramp was used in which mobile phase B increased from 0% at 2 mins to 40% at 72 minutes and
then ramped (1 min) and held constant at 80% (5 min). Initial conditions were reestablished for
the final 11 minutes of the run. The column and trap were further equilibrated at the beginning
of the run using the built-in pre-column (6 µL) and analytical column equilibration (8 µL)
procedures available on the EasyNano LC system. To minimize carryover, blanks (mobile
phase A) were ran between each sample and the sample loop was washed with 1 loop volume
of mobile phase A, 2 volume of mobile phase B, 1 volume of 80% isopropanol, and finally 4
volumes of mobile phase A.
Mass spectrometric analysis was performed using a quadrupole orbitrap (Q-Exactive Plus,
Bremen, Germany) operated in the top 12 data dependent mode. Precursor scans were
performed across a range from m/z 400 to 1400, at a resolving power of 70,000 at m/z 200, an
ion target of 1,000,000, and a max ion injection time of 100 ms. Data dependent MS2 scans
were performed at a resolving power of 12,500 at m/z 200, an ion target of 50,000, and a max
ion injection time of 64 ms. Abundant ions were excluded for 20 seconds, the peptide isotopic
distribution was set to preferred, ion charges of 1,>6 were excluded, and lock mass was
employed using m/z 445.12002.
Data Analysis
Raw files were searched against the SwissProt Reference Mus Musculus database (16657
entries, downloaded 12/13) appended with common protein contaminants using the Sequest HT
algorithm in Proteome Discoverer (version 1.4.0.288, Thermo Fisher Scientific). Data were
searched against a target and decoy database (reversed) within Proteome Discoverer. Search
parameters allowed for up to 2 missed cleavages, carbamidomethylation of cysteines as fixed
modifications, and deamidation of glutamine and asparagine as dynamic modifications.
Precursor and fragment mass measurement accuracy was 10 ppm and 0.6 Da, respectively.
Percolator was used as a post processor to enforce a q value threshold of <0.01. The law of
parsimony was used for protein inference and protein identifications were filtered to those
having a least two unique peptides.
Peptide spectral matches (PSMs) for each protein were summed across the technical
replicates and a Fisher Exact test was performed in Rstudio (V.0.97.551) comparing the
abundances of the CDKs, p21, PCNA in control versus knockout. Further validation of
differential abundance was performed in Skyline by integration of MS1 peak areas. Spectral
libraries were created by importing Proteome Discoverer files directly into Skyline. A
background data was made by loading the mouse fasta file into Skyline and used to check for
peptide uniqueness. Two peptides for each protein were chosen for integration which was
based on uniqueness determined from the background database, no mis-cleavages,
identification in at least one run and abundance via spectral counts. Signals were integrated
and identified as the peptides of interest based on mass measurement accuracy reported by
Skyline, retention time reproducibility, the time at which the MS2 identification occurred
discerned from the spectral library, and the dot product between predicted and observed
isotopic distributions.
Occasionally in the knockout samples, peptide signals from proteins which had low
spectral count could not be confidently integrated and are noted. Peak areas were normalized
to the precursor total ion intensity (TIC) across the 90 minute run and multiplied by 1,000,000 for
visualization purposes.
Quality Control - Statistical Process Control in Proteomics (SProCoP)
A standard albumin digestion (200 fmols) was injected three times prior to the beginning of the
Co-IP analyses to condition the column and establish thresholds to assess analytical variance.
These standards were followed by 2 blank runs. The QC standard was also injected in the
middle of the analyses and at the end. The reproducibility of peak areas, retention time, peak
asymmetry, and mass measurement accuracy of targeted peptides were monitored/evaluated
using the SProCoP algorithm available in Skyline. The CVs for peak areas and RT across the
analyses were less than 20% and 1.5%, respectively. The absolute MMA was < 5 ppm
(external calibration). None of the metrics were outside 2 sd of the mean during the analyses.
Blanks (mobile phase A) were injected after standards.
Supplemental Figures
Figure S1. C/EBPα deficiency results in decreased p21 protein stability following UVB
exposure in the absence of C/EBPα-p21 complex formation. (A) Audioradiography analysis
of p21 in siRNA treated Balb/MK2 keratinocytes pulse labeled with 35S-methionine for 2 hrs
(t=0). Cells were collected at the indicated times after the pulse labeling, and 35S-labeled p21
was immunoprecipitated from cell lysates. Levels of 35S-p21 remaining were determined by
densitometry and plotted to the right. (B) Table of relevant p21 protein-protein interactions
detected by LC-MS/MS, table is representative of 3 separate LC-MS/MS experiments.
Figure S2. Knockdown of Skp2 does not rescue p21 protein levels in C/EBPα depleted
Balb/MK2 cells. Immunoblot analysis of p21, and C/EBPα from nuclear extracts of Balb/MK2
cells treated with combination of control, C/EBPα, and Skp2 siRNAs and collected 6 hrs after
exposure to 5 mJ/cm2 UVB. The total amount of siRNA in each condition was equal.
Knockdown of Skp2 was > 75% as measured by TaqMAN RT-PCR.
gene
siRNA
-
siRNA
-
siRNA
+
siRNA +
siRNA
-
siRNA
-
siRNA +
siRNA +
raw
area
Bio rep1
raw
area
Bio rep2
raw
area
Bio rep1
raw area
n. area
n. area
n. area
n. area
Bio rep2
Bio rep1
Bio rep2
Bio rep1
Bio rep2
K.NLDENGLDLLSK.M
5.6E+08
2.7E+08
3.8E+07
2.0E+07
46.7
39.0
4.9
3.5
R.SPEVLLGSAR.Y
6.2E+08
3.8E+08
3.2E+07
3.5E+07
52.1
56.0
4.2
5.9
R.APEILLGCK.Y
5.8E+08
3.6E+08
4.2E+07
3.6E+07
48.7
52.5
5.5
6.1
K.LLDVIHTENK.L
1.9E+08
1.5E+08
8.8E+06
1.3E+07
16.0
21.5
1.2
2.1
R.VPNGGAAGGGLPVSTVR.E
3.6E+09
1.9E+09
4.6E+08
3.7E+08
297.3
281.9
60.8
62.5
K.VTLVFEHIDQDLR.T
5.5E+09
1.7E+09
3.7E+08
3.6E+08
460.8
247.9
48.3
61.8
R.GSSDVDQLGK.I
1.5E+08
5.7E+07
2.2E+07
2.2E+07
12.8
8.4
2.9
3.7
R.ISAYGALNHPYFQDLER.Y
2.5E+08
1.4E+08
1.6E+07
2.0E+07
20.8
20.7
2.1
3.3
R.WNFDFVTETPLEGNFVWER.V
3.2E+09
1.6E+09
1.5E+08
1.8E+08
271.6
232.8
20.1
30.6
R.DCDALMAGCLQEAR.E
1.7E+09
1.3E+09
1.3E+08
1.4E+08
139.7
190.6
17.5
23.1
R.SEGFDTYR.C
2.9E+08
1.6E+08
ND
ND
24.5
23.2
N/A
N/A
R.YLNFFTK.A
2.7E+08
1.9E+08
ND
ND
22.7
28.2
N/A
N/A
Peptide #1 and Peptide #2
CDK1
CDK2
CDK4
CDK6
P21
PCNA
Table S1. A summary of spectral count data and integrated peak areas for the 6 proteins of
interest. Both raw and normalized peak areas are shown. Spectral count data and peak areas
show similar results.
Figure S3. A second peptide was chosen for each protein to further validate the data and
similar results are shown. Peptide abundances are expressed as a percent of the most
abundant signal for each peptide in a given protein.