Download Detection of Cellular Response to an in vitro Challenge with

Detection of Cellular Response to an in
vitro Challenge with Bacterial Gram-Negative
Lipopolysaccharides (LPS) in Peripheral
Blood Mononuclear Cells (PBMCs) for
Biomarker Research
David Sarracino,1 Jennifer N Sutton,1 Maryann S Vogelsang,1 Bryan Krastins,1 Gregory Byram,1 Amol Prakash,1
Vineet Gupta, 2 Mary F Lopez1
1
Thermo Fisher Scientific, BRIMS (Biomarker Research Initiatives in Mass Spectrometry), Cambridge, MA;
2
Rush University Medical Center, Chicago, Il
Overview
Purpose: To find differentially expressed marker proteins for sepsis in an in vitro
model environment.
Methods: Blood from healthy volunteers is treated with toxic ligands secreted by
gram-negative bacteria. PBMCs are isolated, reduced, alkylated, digested with trypsin.
The resulting peptides are analyzed using liquid chromatography-tandem mass
spectrometry (LC-MS/MS). Differential peptides are identified by Thermo Scientific™
Proteome Discoverer™ software and compared using Thermo Scientific™ Pinpoint™
software.
Results: Full scan quantification of several hundred relevant kinase and pathway
specific proteins generated from over 4000 identified proteins.
Introduction
Gram-negative bacteria, and a major component, lipo-polysaccharides (LPS), are
associated with sepsis. In this study, we look at global protein profiling of mononuclear
cells from LPS-challenged whole blood.
Mononuclear cells are easy to collect and have little of the protein dynamic range
difficulties associated with plasma. In addition, they are responsive to many immune
state conditions, making them ideal targets for biomarker discovery experiments.
Using an in vitro stimulation using a whole blood system directly in tubes used for the
isolation allows for a highly facile method for looking for changes in either secreted
proteins in the plasma fraction or for quick-onset protein changes in the PBMC cell
fraction.
As most gram-negative sepsis infections are from E. coli, we chose the corresponding
LPS. LPS from rod shaped bacteria stimulates the specific Toll like receptor 4(TLR4) in
the MyD88 pathway. Toll like receptors are part of the innate immune response
pathways.
FIGURE 1. MyD88 Pathway
After incubation at 37 ºC for 30
manufacturerʼs instructions for
(~2 mg) were denatured in 350
10 mM Dithiothreitol, reduced/a
and digested overnight with 20
Liquid Chromatography
Peptide retention time standard
plates onto a Thermo Scientific
Thermo Scientific™ Dionex™ P
x 12 cm) connected in a “vente
100 µm x 50 cm packed tip res
Flex™ Ion Source on a hybrid T
Flow and gradient from 4-50%B
0.2% formic acid, water(v/v). Bu
0.2% formic acid (v/v), all solve
Portions of each of the TLR4 di
were fractionated into 12 fractio
8 µm particle 300A pore, buffer
hydroxide, Buffer B: 29 mM am
using a flow rate of 1 mL/min in
Mass Spectrometry
For mass spectrometric analys
Full MS scans acquired at a res
dependent scans analyzed in th
Uncharacterized charge states
phase triggering with monoisoto
minimum peak threshold of 3.5
was set to 100 ms. Dynamic ex
Data Analysis
Full-scan comparisons were ma
processed by Proteome Discov
different peptide identification s
3) only searches for high-confid
complex search strategy (Figur
nodes, where small groups of P
in each node. This allows for h
of each database, albeit at an i
information was processed usin
shown). Pinpoint software allow
obtained from data from both u
chromatography in all samples
FIGURE 2. High pH reverse
phase fractionation for librar
peptide fractionation
Cascades in this pathway involve many signaling events that are either proteolytic
cleavages, phosphorylations or other modifications. The large number of human
proteins and their associated post-translational modifications (PTM) represent a
challenge for MS-based biomarker discovery. To this effect, the simplest sample
preparation techniques will provide the most reproducible results.
Methods
Sample Preparation
Blood samples from a healthy single donor were collected into BD Vacutainer™ CPT
Cell Separation Tubes (Becton Dickinson) in accordance with IRB approval. Buffers
and stimulant solutions were injected directly into the blood collection tubes using a
1 mL syringe with a 27 ga needle. Control tubes had 200 µL of phosphate buffered
saline added and were prepared in parallel to the stimulated tubes. LPS-EB Toll Like
Receptor 4 Ligand (InvivoGen, San Diego, CA) was added to a concentration of
100 ng/mL (Low stim) and 10 µg/mL (High stim) of whole blood.
FIGURE 3. Search
workflow for protein
phosphorylation.
2 Detection of Cellular Response to an in vitro Challenge with Bacterial Gram-Negative Lipopolysaccharides (LPS) in Peripheral Blood Mononuclear Cells (PBMCs)
eins for sepsis in an in vitro
h toxic ligands secreted by
, alkylated, digested with trypsin.
matography-tandem mass
dentified by Thermo Scientific™
Thermo Scientific™ Pinpoint™
elevant kinase and pathway
proteins.
-polysaccharides (LPS), are
l protein profiling of mononuclear
f the protein dynamic range
re responsive to many immune
ker discovery experiments.
m directly in tubes used for the
or changes in either secreted
in changes in the PBMC cell
oli, we chose the corresponding
ecific Toll like receptor 4(TLR4) in
innate immune response
After incubation at 37 ºC for 30 min the cells were isolated according to the
manufacturerʼs instructions for a total exposure time of 60 min. Rinsed cell pellets
(~2 mg) were denatured in 350 µL of 8M Urea 300 mM Tris-HCl 2.5% n-propanol
10 mM Dithiothreitol, reduced/alkylated, diluted to 2 mL with 50 mM Tris, 5 mM CaCl2
and digested overnight with 20 µg of Pierce Trypsin Protease, MS Grade.
Liquid Chromatography
Peptide retention time standards were added and the samples were loaded into 96-well
plates onto a Thermo Scientific™ EASY-nLC™ 1000. Separations were done on a
Thermo Scientific™ Dionex™ PS-DVB trap column, (5 µm particle, 300Å pore, 150 µm
x 12 cm) connected in a “vented t” configuration to a 5 µm particle, 200Å pore C18AQ
100 µm x 50 cm packed tip resolving column in a Thermo Scientific™ Nanospray
Flex™ Ion Source on a hybrid Thermo Scientific™ Orbitrap Velos Pro™ MS. Stepped
Flow and gradient from 4-50%B at 650 nL/min over 205 min. Buffer A is 2% Methanol
0.2% formic acid, water(v/v). Buffer B is 10% water, 10% isopropanol, 80% acetonitrile
0.2% formic acid (v/v), all solvents are Thermo Scientific™ Optima™ LC-MS grade.
Portions of each of the TLR4 digests (Low and High stim) pooled for library creation
were fractionated into 12 fractions of 1.8 ml, on a 4.6 mm x 25 cm PS-DVB column
8 µm particle 300A pore, buffer A: 100 mM ammonium formate, 58 mM ammonium
hydroxide, Buffer B: 29 mM ammonium hydroxide in 91% acetonitrile 9% water (v/v),
using a flow rate of 1 mL/min in a gradient to 45% B (Figure 2).
Mass Spectrometry
For mass spectrometric analysis, a data-dependent top 25 method has been used .
Full MS scans acquired at a resolution of 100,000 using a 1e6 target value, with
dependent scans analyzed in the linear ion trap with normal scan resolution.
Uncharacterized charge states and + 1 charge states are rejected. Chromatography
phase triggering with monoisotopic fitting was used with a peak width of 40 s and a
minimum peak threshold of 3.5e4. The maximum inject time allowed for MS/MS scans
was set to 100 ms. Dynamic exclusion is turned on using a peak width of 60 s.
FIGURE 5. Results from different
then be brought into Pinpoint soft
Data Analysis
Full-scan comparisons were made using Pinpoint software, and MS/MS spectra were
processed by Proteome Discoverer software using The Mascot® search engine. Two
different peptide identification strategies were used. The simple search method (Figure
3) only searches for high-confidence, tryptic peptides and phosphopeptides. The more
complex search strategy (Figure 4), breaks the PTM search strategy into multiple
nodes, where small groups of PTMs, likely to occur on the same peptide, are searched
in each node. This allows for higher-confidence assignments due to the reduced size
of each database, albeit at an increased search computational time. Pathway
information was processed using Thermo Scientific™ ProteinCenter™ software (not
shown). Pinpoint software allows for the import of spectral libraries which can be
obtained from data from both unfractionated and fractionated samples provided the
chromatography in all samples is reproducible and retention times are consistent.
FIGURE 2. High pH reverse
phase fractionation for library
peptide fractionation
nts that are either proteolytic
he large number of human
cations (PTM) represent a
effect, the simplest sample
ible results.
cted into BD Vacutainer™ CPT
ce with IRB approval. Buffers
blood collection tubes using a
200 µL of phosphate buffered
ulated tubes. LPS-EB Toll Like
dded to a concentration of
ole blood.
FIGURE 4. Search workflow for mu
into groups of most likely to occu
computationally intensive and wo
analyzed MS2. Many modifications
deamidations, semi-tryptic, and di
other search engines without com
Results
FIGURE 3. Search
workflow for protein
phosphorylation.
In order to allow the detection of diffe
instrumentation should provide enou
simultaneously providing MS/MS frag
peptides as possible. In this experim
confidence containing >250 phospho
oxidations (other than methionine) th
Pinpoint software has the advantage
library import. In this example, all kin
analysis. Once selected, proteins tha
verified using Pinpoint software (Figu
specific proteins of interest (Figure 8
(Figure9) MAPKKK 15, can be selec
Thermo Scientific Poster Note • PN ASMS13_W457_DSarracino_E 07/13S 3
e isolated according to the
me of 60 min. Rinsed cell pellets
0 mM Tris-HCl 2.5% n-propanol
o 2 mL with 50 mM Tris, 5 mM CaCl2
sin Protease, MS Grade.
FIGURE 4. Search workflow for multiple modifications. Searches are broken up
into groups of most likely to occur modifications. This search strategy is
computationally intensive and works best with the high-resolution Orbitrapanalyzed MS2. Many modifications such as ubiquitination, oxidations and
deamidations, semi-tryptic, and different databases can be searched even by
other search engines without compromising the integrity of the results.
FIGURE 6. Quantification of th
GDDTPLHLAASHGHR from in
sapiens] A) Comparison of Co
all isotopes.
A
the samples were loaded into 96-well
000. Separations were done on a
mn, (5 µm particle, 300Å pore, 150 µm
o a 5 µm particle, 200Å pore C18AQ
Thermo Scientific™ Nanospray
™ Orbitrap Velos Pro™ MS. Stepped
er 205 min. Buffer A is 2% Methanol
er, 10% isopropanol, 80% acetonitrile
cientific™ Optima™ LC-MS grade.
gh stim) pooled for library creation
4.6 mm x 25 cm PS-DVB column
nium formate, 58 mM ammonium
e in 91% acetonitrile 9% water (v/v),
% B (Figure 2).
ent top 25 method has been used .
0 using a 1e6 target value, with
with normal scan resolution.
ates are rejected. Chromatography
ed with a peak width of 40 s and a
inject time allowed for MS/MS scans
on using a peak width of 60 s.
FIGURE 5. Results from different search strategies and any fractionation can
then be brought into Pinpoint software through the spectral library function.
FIGURE 7. View of the replicat
isotopes (792.387, green: 792.
Control, Low and High stimula
software, and MS/MS spectra were
g The Mascot® search engine. Two
ed. The simple search method (Figure
des and phosphopeptides. The more
TM search strategy into multiple
ur on the same peptide, are searched
assignments due to the reduced size
omputational time. Pathway
ic™ ProteinCenter™ software (not
spectral libraries which can be
fractionated samples provided the
d retention times are consistent.
FIGURE 8. Signal Pathway pro
GGEIEGFR from transforming
[Homo sapiens].
Results
In order to allow the detection of differentially expressed proteins and peptides,
instrumentation should provide enough quantitative full-scan measurements while
simultaneously providing MS/MS fragmentation data to allow sequencing of as many
peptides as possible. In this experiment, ca 4000 proteins were identified over 95%
confidence containing >250 phosphorylations, >150 ubiquitinylations, peptide
oxidations (other than methionine) that were inserted into the Pinpoint spectral library.
Pinpoint software has the advantage in that it allows for a selected protein/peptide
library import. In this example, all kinases identified in the library can be selected for
analysis. Once selected, proteins that are up or down regulated can be highlighted and
verified using Pinpoint software (Figures 6, 7). In addition to protein class selection,
specific proteins of interest (Figure 8) TGF beta and pathways specific components,
(Figure9) MAPKKK 15, can be selected and analyzed for quantification.
4 Detection of Cellular Response to an in vitro Challenge with Bacterial Gram-Negative Lipopolysaccharides (LPS) in Peripheral Blood Mononuclear Cells (PBMCs)
fications. Searches are broken up
ons. This search strategy is
h the high-resolution Orbitrapbiquitination, oxidations and
bases can be searched even by
he integrity of the results.
egies and any fractionation can
gh the spectral library function.
ressed proteins and peptides,
ve full-scan measurements while
ata to allow sequencing of as many
0 proteins were identified over 95%
50 ubiquitinylations, peptide
rted into the Pinpoint spectral library.
ws for a selected protein/peptide
ed in the library can be selected for
own regulated can be highlighted and
addition to protein class selection,
nd pathways specific components,
yzed for quantification.
FIGURE 6. Quantification of the +2 charge state of peptide
GDDTPLHLAASHGHR from integrin-linked protein kinase gi|4758606 [Homo
sapiens] A) Comparison of Control, Low and High Stimulation B) Alignment of
all isotopes.
A
FIGURE 9. MyD88 signal path
Peptide QILEGLK from mitog
gi|282847398 [Homo sapiens]
B
FIGURE 7. View of the replicates of sample quantifications from the first two
isotopes (792.387, green: 792.889, blue) of the peptide GDDTPLHLAASHGHR in
Control, Low and High stimulation experiments (pairs, left to right).
FIGURE 8. Signal Pathway proteins up-regulated from LPS stimulation. Peptide
GGEIEGFR from transforming growth factor beta-1 precursor gi|63025222
[Homo sapiens].
Conclusion

The in vitro whole blood
sample preparation wo
method of testing cell s
samples.

Spectral Library creatio
large number of differen
enrichment strategy.

Pinpoint software allow
modes in complex sam

Future work will be to u
list of peptides for selec
Thermo Scientific™ TS
instruments. These wil
difficult to quantify in th
pathways that were sho
are also correlated with
References
1.
Angus DC, Linde-Zwirb
(29), 1303-1310
2.
Robbins Basic Patholog
3.
Mendes ME, Baggio-Za
Salomao R. Immunobio
Mascot is a registered trademark of Matri
trademarks are the property of Thermo Fi
This information is not intended to encour
intellectual property rights of others.
For Research Use Only. Not for use in di
Thermo Scientific Poster Note • PN ASMS13_W457_DSarracino_E 07/13S 5
ate of peptide
rotein kinase gi|4758606 [Homo
High Stimulation B) Alignment of
FIGURE 9. MyD88 signal pathway proteins up-regulated from LPS stimulation.
Peptide QILEGLK from mitogen-activated protein kinase kinase kinase 15
gi|282847398 [Homo sapiens].
B
quantifications from the first two
he peptide GDDTPLHLAASHGHR in
nts (pairs, left to right).
ated from LPS stimulation. Peptide
beta-1 precursor gi|63025222
Conclusion

The in vitro whole blood PBMC stimulation model combined with a simple
sample preparation workflow strategy allows for a facile and reproducible
method of testing cell signaling in the immune response from human research
samples.

Spectral Library creation from simple fractionation techniques allows for a
large number of different PTMs to be identified without using any specific PTM
enrichment strategy.

Pinpoint software allows for targeted quantification in even full scan discovery
modes in complex sample mixtures.

Future work will be to utilize the library and initial data to generate a targeted
list of peptides for selected reaction monitoring (SRM) workflows on both
Thermo Scientific™ TSQ Vantage™ and Thermo Scientific™ Q Exactive™
instruments. These will be to address the low level library peptides that were
difficult to quantify in this workflow, but are of interest in the biological
pathways that were shown to be up- and down-regulated in these samples and
are also correlated with sepsis.
References
1.
Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Critical Care Med 2001,
(29), 1303-1310
2.
Robbins Basic Pathology. 8th edition
3.
Mendes ME, Baggio-Zappia GL, Brunialti MK, Fernandes Mda L, Rapozo MM,
Salomao R. Immunobiology, 2011 Mar;216(3):285-95.
Mascot is a registered trademark of Matrix Science. Vacutainer is a trademark of Becton Dickinson. All other
trademarks are the property of Thermo Fisher Scientific and its subsidiaries
This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
For Research Use Only. Not for use in diagnostic procedures.
6 Detection of Cellular Response to an in vitro Challenge with Bacterial Gram-Negative Lipopolysaccharides (LPS) in Peripheral Blood Mononuclear Cells (PBMCs)
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