Profiling of the Human Monocytic Cell Secretome by Quantitative Label-free Mass Spectrometry
Identifies Stimulus-specific Cytokines and Proinflammatory Proteins
M. Groessl1, H. Luksch2, A. Rösen-Wolff2, A. Shevchenko1, M. Gentzel1
1
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
2
Department of Pediatrics, University Medical Center Carl Gustav Carus, Dresden, Germany
Supplementary Information
Figure S1. Correlation plots of normalized protein intensities of quantified proteins (NI; a.u.:
arbitrary units) for biological replicates (average of two technical replicates) between samples of the
control group. (A) Correlation of Experiment #1 to Experiment #2 (B) Correlation of Experiment #1 to
Experiment #3 (C) Correlation of Experiment #2 to Experiment #3.
Figure S2 Correlation plots of normalized protein intensities of quantified proteins (NI; a.u.: arbitrary
units) for biological replicates (average of two technical replicates) between samples of the LPS/ATP
group. (A) Correlation of Experiment #1 to Experiment #2 (B) Correlation of Experiment #1 to
Experiment #3 (C) Correlation of Experiment #2 to Experiment #3.
Figure S3. Correlation plots of normalized protein intensities of quantified proteins (NI; a.u.:
arbitrary units) for biological replicates (average of two technical replicates) between samples of the
LPS/MDP group. (A) Correlation of Experiment #1 to Experiment #2 (B) Correlation of Experiment #1
to Experiment #3 (C) Correlation of Experiment #2 to Experiment #3.
Figure S4. Correlation plots of normalized peptide intensities of quantified peptides (NI; a.u.:
arbitrary units) for technical replicates between samples of the control group (Con, first row,
n=4082), LPS/ATP group, (second row, ATP, n=5508) and LPS/MDP group (third row, MDP, n=4560).
First column: Correlation of technical replicates of experiment #1, second column: Correlation of
technical replicates of Experiment #2, third column: Correlation of technical replicates of Experiment
#3.
Figure S5. Correlation plots of normalized peptide intensities of quantified peptides (NI; a.u.:
arbitrary units) for biological replicates (average of two technical replicates) between samples of the
control group. (Con, first row, n=4082), LPS/ATP group, (second row, ATP, n=5508) and LPS/MDP
group (third row, MDP, n=4560). First column: Correlation of biological replicates of experiment #1
and #2, second column: Correlation of biological replicates of Experiment #2 and #3, third column:
Correlation of biological replicates of Experiment #1 and #3.
Figure S6. Correlation plots of normalized protein intensities of quantified proteins (NI; a.u.:
arbitrary units) for technical replicates between samples of the control group (Con, first row, n=561),
LPS/ATP group, (second row, ATP, n=724) and LPS/MDP group (third row, MDP, n=635). First
column: Correlation of technical replicates of experiment #1, second column: Correlation of
technical replicates of Experiment #2, third column: Correlation of technical replicates of Experiment
#3.
Table S1. Recovery of selected cytokines after ultrafiltration determined by CBA assay
1
3245
3158
60
IL-1β
2
2239
2185
53
Recovery Concentrate (%)
97.3
Recovery Flow-Through (%) 1.9
97.6
2.4
Sample #
Supernatant (pg/ml)
Concentrate (pg/ml)
Flow-Through (pg/ml)
IL-8
TNF
3
1
2
3
1
2
3075 20095 20404 18347 1138 916
2956 19683 19789 17488 1112 887
62
101
99
126
12
11
3
1523
1457
17
96.1
2.0
95.7
1.1
97.9
0.5
97.0
0.5
95.3
0.7
97.7
1.0
96.8
1.2
Table S2. Settings used for ionisation and mass spectrometric detection (Triversa Nanomate and
Orbitrap LTQ Velos).
Setting
Ionisation voltage
Gas pressure
Scan range
Nominal resolution (at m/z 400)
Lock mass
Data mode
Activation type
Intensity threshold for DDA
Isolation width
Normalized collision energy
Activation Q
Activation Time
MS/MS scans per precursor scan
Dynamic exclusion list size
Dynamic exclusion duration
Exclusion mass width relative to mass
Value
1.7 kV
0.45 psi
380 – 1200 m/z
60000
445.120025 m/z (Siloxane)
Centroid
CID
2000
2 m/z
35 %
0.25
30 ms
6
500
300 s
6 ppm
Table S3. Mascot Settings
Setting
Peptide tolerance
MS/MS tolerance
Enzyme
Fixed modifications
Variable modifications
Max. missed cleavages
Value
5 ppm
0.6 Da
Trypsin
Carbamidomethyl (C)
Oxidation (M)
2
Table S4. Settings used for peptide validation and protein assembly in the Transproteomic Pipeline.
Setting
Analysis Pipeline
Enzyme
Minimum peptide length
Probability filter (peptide- and protein-level)
Run Peptide Prophet
Use icat information
Do not Use icat information
Use N-glyc motif information
Use pI information
Use Hydrophobicity / RT information
Use Phospho information
Accurate mass binning
Do not use the NTT model
Do not use the NMC model
MALDI data
Exclude all entries with asterisked score values
Leave alone all entries with asterisked score values
Run iProphet
Run ProteinProphet
Include zero probability proteins
Value
Mascot
Trypsin
7
0.05
yes
no
no
no
no
no
no
yes
no
no
no
no
no
yes
yes
no
Table S5. Settings used for feature detection in PVIEW.
Setting
Quantification Mode
Load MS/MS spectra
Run processing algorithms
Peak threshold
XIC delta time
XIC delta
XIC min length
XIC max length
Isotope tolerance
Align translation
Align nonlinear
XIC width
Group delta time
Minimum conditions
Minimum replicate sets
Value
label-free time alignment
yes
yes
0
240 s
5 ppm
15 s
300 s
3.5 ppm
yes
yes
5 ppm
120 s
1
3
Table S6. DanteR workflow
Step
1
2
3
4
5
Action
Grouping of single LC-MS runs (type of stimulation; technical and biological
replicates)
log2 transformation of peptide intensities
Protein level one-way ANOVA (linear model; min. 2, max. 5 peptides; technical and
biological replicates as factors; control group as first factor reference level)
p-value adjustment (Benjamini-Hochberg FDR correction)
Split significant proteins (up or down regulation; 2-fold change; p<0.01)
Table S7. Peptides with corresponding m/z values used for quantification of selected cytokines.
Protein
IL-1β
Peptides
NLYLSCVLKDDKPTLQLESVDPK
CSFQDLDLCPLDGGIQLR
m/z
892.4715
1054.0014
IL-8
ELCLDPKENWVQR
VIESGPHCANTEIIVK
843.9166
883.9601
TNFα
VNLLSAIK
TPSDKPVAHVVANPQAEGQLQWLNR
DNQLVVPSEGLYLIYSQVLFK
429.2768
919.1475
809.1056
CCL3
SRQVCADPSEEWVQK
QIPQNFIADYFETSSQCSKPGVIFLTK
606.9527
780.3950
CCL3L1
GRQVCADPSEEWVQK
QIPQNFIADYFETSSQCSKPSVIFLTK
894.9208
787.8974
PTX3
LTSALDELLQATR
ALAAVLEELR
ADLHAVQGWAAR
LAESLARPCAPGAPAEAR
SWLPAGCETAILFPMR
715.8961
542.8213
432.2262
612.9843
924.9609
DOPD
FFPLESWQIGK
PFLELDTNLPANRVPAGLEK
SHSAHFFEFLTK
676.3565
732.0671
725.8588
Table S8. Concentration of selected cytokines in supernatants of cells cultured with supplementation
of 10% FCS or under serum-free conditions (determined by CBA assay; all values in pg/ml).
Control
LPS/ATP
LPS/MDP
10% FCS Serum-free
10% FCS
Serum-free
10% FCS
Serum-free
IL-1β 290±70
18±10
3709±1177
2853±538
380±77
233±26
IL-8 230±58
68±26
22232±4771 19616±1109 7314±1041 5621±442
TNF
49±6
15±10
2158±611
1192±307
1110±375
695±245
Table S9. Total protein contents of different sample groups (n=3) determined by BCA assay.
Control
LPS/ATP
LPS/MDP
Total Protein (ug/ml)
14.5±2.0
18.4±2.7
15.7±1.9