Download The Oxidized Proteome of Peripheral Blood Mononuclear Cells: A

demonstrated that the combination of the results yielded the identification of over 8000
proteins. In addition, over 5000 proteins were accurately quantified and over 7000
oxidation events were identified. Remarkably, our data also suggest that H2O2 might play
a role modulating signaling pathways by reacting with specific protein targets. Overall, this
study not only adds significant value in the mechanistic understanding of redox signaling,
but it also creates a valuable protein repository that could lead to the development of new
Poster Note 644 8 7
The Oxidized Proteome of Peripheral
Introduction
Blood Mononuclear Cells:
A Valuable
Repository for Clinical Proteomics
THE OXIDIZED PROTEOME OF PERIPHERAL BLOOD MONONUCLE
Re
Xiaolei Xie , Xiaoyue Jiang , Monica Carrera , Daniel Lopez-Ferrer , Andreas FR Huhmer
Thermo Fisher Scientific, San Jose, USA; Spanish National Research Council (CSIC),
A VALUABLE
REPOSITORY FOR CLINICAL PROTEOMICS
Vigo, Spain
therapeutic strategies.
Peripheral blood mononuclear cells (PBMCs) are a popular model system to study the
physiological and metabolic activity of cells within the body. PBMCs have enabled a very
1
1
1
2
2
broad collection of biomedical applications. Monitoring gene expression and
1
1
posttranslational modifications are very promising areas in biomarker discovery and
FIG
pe
we
translational research. In this study, we have aimed to have the most extensive proteome
map of PBMCs and monitor the in vitro effect of reactive peroxide at low concentration
2 Daniel
1, mapped,
under, different
exposure
times. Over 8000 proteins were
more than 5000
Xiaolei Xie1, Xiaoyue Jiang1, Monica Carrera
Lopez-Ferrer
Andreas
FR Huhm
proteins were accurately quantified and over 7000 oxidation events were identified. These
1Thermo Fisher Scientific, San Jose, USA, 2Spanish National Research Council (CSIC)
observations represent the largest proteome profiling dataset for PBMCs to date, and
create useful warehouse in the clinical blood proteomics field.
Overview
Purpose: Peripheral blood mononuclear cells (PBMCs) and its specific cell subsets have
allowed for a very broad collection of applications such as in vitro cell-based assays to
study the immune response to a given stimuli, or to monitor in vivo or ex vivo changes
before and after a drug treatment. More importantly, they are an easily accessible cellular
part of the blood, and they are the only component of the blood that could have a gene
expression activity. Access to complete atlas of the gene expression and posttranslational
Study
Design
Methods
Sample Preparation
PBMCs from a healthy male individual were purchased from AllCells. 1mill cells aliquots
were in vitro treated with 5 mM H2O2 for 0, 2, 10, 30 and 80 min. Cell lysis, protein
precipitation and digestion were performed using the Mass Spec Sample Prep Kit for
Cultured Cells (Pierce, Rockford IL).
modifications in PBMCs will permit more sophisticated studies such as the selection of
Liquid Chromatography and Mass Spectrometry Analsys
potential biomarkers that could be used for many purposes ranging from diagnostic,
Peptide digests were then analyzed by LC-MS/MS analysis on a Easy nLC1000 coupled
prognosis or even help selecting the appropriate therapy for a patient.
to a Thermo ScientificTM Q ExactiveTM Plus mass spectrometer over a 2-hour gradient.
Results: This study compiles the most extensive proteome map of PBMCs. We have
Data Analysis
demonstrated that the combination of the results yielded the identification of over 8000
proteins. In addition, over 5000 proteins were accurately quantified and over 7000
oxidation events were identified. Remarkably, our data also suggest that H2O2 might play
a role modulating signaling pathways by reacting with specific protein targets. Overall, this
study not only adds significant value in the mechanistic understanding of redox signaling,
but it also creates a valuable protein repository that could lead to the development of new
DONOR
Peripheral blood mononuclear cells (PBMCs) are a popular model system to study the
physiological and metabolic activity of cells within the body. PBMCs have enabled a very
broad collection of biomedical applications. Monitoring gene expression and
posttranslational modifications are very promising areas in biomarker discovery and
translational research. In this study, we have aimed to have the most extensive proteome
map of PBMCs and monitor the in vitro effect of reactive peroxide at low concentration
COLLECT PBMC
phosphor RS. These tools were used as nodes within Thermo ScientificTM Protein
FIG
Discoverer 2.0 software. Inferno was then used for further statistical analysis and
ProteinCenter was used to extract biological context and set comparisons with publicly
pro
available datasets.
de
SAMPLE PREPARATION
LC-MS ANALYSIS
Q Exactive Plus MS
FIGURE 1. Workflow summarizing the analytical approach. Samples were obtained from a donor,
performed, proteins precipitated and digested. After digestion, the samples were acidified, dried down
well as, label free quantitation. Subsequently, statistics and data mining including gene ontology enric
Results
Control
A
under different exposure times. Over 8000 proteins were mapped, more than 5000
B
10 min
proteins were accurately quantified and over 7000 oxidation events were identified. These
observations represent the largest proteome profiling dataset for PBMCs to date, and
30 min
create useful warehouse in the clinical blood proteomics field.
Methods
Sample Preparation
CENTRIFUGATION
Database search and oxidation site localization were performed using SEQUEST and
therapeutic strategies.
Introduction
BLOOD EXTRACTION
80 min
40453
PBMCs from a healthy male individual were purchased from AllCells. 1mill cells aliquots
were in vitro treated with 5 mM H2O2 for 0, 2, 10, 30 and 80 min. Cell lysis, protein
precipitation and digestion were performed using the Mass Spec Sample Prep Kit for
80 min
1M H2O2
ID
D
Cultured Cells (Pierce, Rockford IL).
Liquid Chromatography and Mass Spectrometry Analsys
Peptide digests were then analyzed by LC-MS/MS analysis on a Easy nLC1000 coupled
to a Thermo ScientificTM Q ExactiveTM Plus mass spectrometer over a 2-hour gradient.
Data Analysis
Database search and oxidation site localization were performed using SEQUEST and
phosphor RS. These tools were used as nodes within Thermo ScientificTM Protein
UNIQUE IDENTIFIED PEPTIDES
METH
Discoverer 2.0 software. Inferno was then used for further statistical analysis and
FIGURE 2. In-depth coverage of the PBMCs proteome. A. Number of unique identified peptides ga
ProteinCenter was used to extract biological context and set comparisons with publicly
proteins with oxidized cysteines and/or methionine. C. Venn diagram representing the frequency of pr
USA, 2Spanish National Research Council (CSIC), Vigo, Spain
Study Design
Replicates
e
onal
DONOR
BLOOD EXTRACTION
CENTRIFUGATION
COLLECT PBMC
30 min
5 mM
H2O2
10 min
80 min
80 min
TREATMENT
ular
0 min
1M
H2O2
4 TIME POINTS
ave
F
0
i
a
ay
b
this
t
ng,
new
ery
me
SAMPLE PREPARATION
LC-MS ANALYSIS
Q Exactive Plus MS
IDENTIFICATION, ANNOTATION &
INTERPRETATION
PBMCs REDOXOME
ATLAS
FIGURE 1. Workflow summarizing the analytical approach. Samples were obtained from a donor, slow centrifuged to separate plasma and erythrocytes from PBMCs. Lysis was then
performed, proteins precipitated and digested. After digestion, the samples were acidified, dried down and analyzed by LC-MS. Finally, peptide and protein identification was performed, as
well as, label free quantitation. Subsequently, statistics and data mining including gene ontology enrichment was performed.
Results
Control
A
B
10 min
ese
Replicate 1
Replicate 2
6% Oxidized Proteins
C
F
9895
12611
p
PROTEINS
c
30 min
Replicate 3
80 min
40453
ts
80 min
1M H2O2
IDENTIFIED PROTEINS
D
E
1762
ed
CORE PROTEOME
.
12% Oxidation
7% Oxidation
1.4% Dioxidation
91% Trioxidation
F
793
88% Dioxidation
UNIQUE IDENTIFIED PEPTIDES
METHIONINE OXDATION SITES
T
C
CYSTEINE OXIDATION SITES
FIGURE 2. In-depth coverage of the PBMCs proteome. A. Number of unique identified peptides gained after the run for each condition. B. Number of identified proteins and number of
proteins with oxidized cysteines and/or methionine. C. Venn diagram representing the frequency of protein identification within the three replicates. Proteins identified in all 3 replicates were
designated as core proteome. D and E. Number of unique oxidation sites for methionine and cytsteine, and percentage of the different oxidation states identified within each amino acid
2 The Oxidized Proteome of Peripheral Blood Mononuclear Cells: A Valuable Repository for Clinical Proteomics
d
10 min
30 min
80 min
PC3 (5.5%)
0 min
PC1 (64.4%)
CORRELATION
PLOTS
LOG TRANSFORMED
INTENSITIES
LOWES NORMALIZED
INTENSITIES
PCA PLOT
FIGURE 3. Statistical Analysis Workflow. Areas from the identified peptides were extracted using the Precursor Ions Area Detector plug-in in Proteome Discoverer software and exported
in to a text file for further analysis. Reproducibility was first evaluated using a correlation plot to discard outliers. The average correlation was 0.9. Then raw intensities were log2 transformed
and normalized using LOWES. Peptides were roll-up to protein as follows. A reference peptide with the most presence across all the datasets, was chosen from the group of peptides that
belong to a protein. Then the ratios of peptide abundances with respect to the reference were computed, and their median was used as a scaling factor. Protein abundance was obtained as
the median of the resulting peptide abundances. Finally, PCA and ANOVA test were performed to classify the samples and discover those protein that changed in abundance.
A
B
1
0
10
30
80
80_1M
0
10
30
80
80_1M
Figure 6.Differential protein expression upon oxidative stress treatment for the whole proteome. Differentially, expressed proteins (p-value<0.01) were analyzed by KEGG.db
package after the extraction of the corresponding genes. The top functional identifiers mapped to (A) the Antigen and Presentation signaling pathway in T-cells, as well as to (B) the TCA
cycle pathway (red stars). Heatmaps represent the normalized abundance for the significant proteins that map to those pathways.
tion
0min
10min
30min
80min
0min
80min_1M
10min
30min
80min
80min_1M
Figure 7. Time course profiles of the oxidation sites. Groups of oxidized proteins (query genes) with similar dynamic behaviors were clustered using k-means method. Euclidian
distances were calculated for a total of 9 different clusters. Two clusters with opposite profiles are exemplified. In addition, pathway enrichment analyses were performed for each cluster.
The analysis demonstrates that both the immunological response and TCA cycle reaction pathways were among the most affected in PBMCs after an oxidative stress treatment.
Conclusion

Simple, but yet powerful proteomic workflow, based on label free quantitation, single UHPLC runs on a bench top mass spectrometer and data analysis by Proteome Discoverer
software.

Largest coverage of the PBMC proteome and its dynamics to oxidative stress1.
1 Saša Končarević, et al, “In-Depth Profiling of the Peripheral Blood Mononuclear Cells Proteome for Clinical Blood Proteomics,”International Journal of Proteomics, vol. 2014, Article ID 129259 doi:10.1155/2014/129259
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