Download Proteomic Strategies to Analyze Cell

PROTEOMIC STRATEGIES TO ANALYZE CELL-FREE FRACTIONS
FROM ACTIVATED PERIPHERAL BLOOD MONONUCLEAR
CULTURES
S. D'Costa, J. Wilkinson, C. Aparicio, E. Rabellino, and W. Bolton
Custom BioPharma Solutions, Beckman Coulter, Inc.
• Evaluate the proteome profile of activated PBMC
supernatants
• Finer mapping of secreted proteome signatures
using strategies to deplete the most abundant
proteins in culture medium containing serum.
Injector
Chromatofocusing
Column
Cell-Free Supernatants
Reverse
Phase
Column
Un fractionated
Supernatants
Un fractionated
Supernatants
Automated
liquid
handler
Run 0.125 ml of
Supernatants
Through IgY
Fractionation
Column (10 Runs)
Increasing pI
pH monitor
•
Orosomucoid
IgA
IgM
Alpha 2-Macroglobulin
HDL (apo A-I)
IgG
HDL (apo A-II)
Fibrinogen
Alpha 1-ATT
Haptoglobin
Transferrin
Control IgY sups
Activated IgY sups
PH gradient
8.5Æ4>0
0.010
0.005
Absorbance at 280nm
Absorbance
0.05
• The IgY fractionation procedure enables a highly reproducible
separation of the most abundant proteins in human serum by
affinity chromatography.
IgY fractionated
Non fractionated
• The Igy fractionated supernatant enables the identification of
proteins of lower concentration that are possibly “masked” by
the higher concentration serum proteins
0.03
0.02
• The IgY fractionation procedure enables the identification of
cell secreted components that are associated with high
abundant proteins in serum
minutes
0.01
0.00
50
Run in minutes
Complete Proteome Profile of
Activated Supernatants
Comparison of IgY Fractionated
Control and Activated supernatants
Flow through
0.000
35
Increasing pI
Heartfelt thanks to Dr. Gupta, Dr. Rau, Hull, O’connell and
Bloodgood for their technical assistance
• Qualitative and quantitative differences and similarities of
protein profiles in control and activated supernatants are
easily observed
High Salt Wash
0.020
0.015
OBSERVATIONS AND CONCLUSIONS
• Complete proteome profiles of secreted components from
control and activated PBMCs are easily obtained and evaluated
using the PF2D gel-free separation system.
0.07
Bound
0.04
25
IgY Fractionated
Reversed phase
First Dimension Profile of IgY Fractionated Supernatants
Compared to Non-Fractionated Supernatants
Flow Through
0
0.025
difference
Increasing pI
0.06
Reproducibility of Fractions
Through the column
activated
Chromatofocussing
Completely automated platform of two-dimensional
separation of complex mixtures in a liquid and intact
state.
*ProteomeLab IgY LC10 Kit A24355-Beckman Coulter Inc.
Control IgY sups RUN 7------Control IgY sups RUN 8------Control IgY sups RUN 9-------
Comparison of Activated and IgY Fractionated Activated
Supernatants Using DeltaVue-Differential Display Software
UV Detector
2nd Dimension: Further separation of each pI range liquid
fraction based on hydrophobicity
Proteins Depleted Using IgY 12 Primate Affinity Column*
HAS
UV Detector Fraction Collector/
Injector
1st Dimension: Separation of proteins into distinct pI
range liquid fractions
•
mins
control
Fraction
Collector
Cell-Free Supernatants
•Concentrate supernatants using Millipore 5kDa cutoff filtration device
•Run Concentrated Supernatants on PF2D Platform
0
difference
SEB/CD28 Activated
Leave at 37 Deg C for 24 hrs
Run 0.125 ml of
Supernatants
Through IgY
Fractionation
Column (10 Runs)
activated
2nd Dimension
Increasing Hydrophobicity
Control Un-activated
1st Dimension
Comparison of 2-dimensional Fractionated Control and Activated
Supernatants Using DeltaVue-Differential Display Software
• Majority of the proteins in both control and activated cultures
are eluted at lower pH ranges in the pH gradient
Increasing Hydrophobicity
AIM
Fresh-Ficoll Purified PBMCs
Absorbance at 280nm
The use of Proteomic strategies in the discovery process is
imperative since post-transcriptional modification can
produce dramatic changes in protein levels and activity that
are invisible to DNA arrays. The introduction of new and
improved proteomics solutions with increased sensitivity,
specificity and ease of use has been integral in facilitating
this process. The current study has evaluated signatures of
immune response in cell-free fractions of control and
activated peripheral blood nuclear cell cultures using
proteomic combinations designed to improve sensitivity of
detection and ease of use.
Peripheral blood mononuclear cells cultured in medium
containing human AB serum were subjected to activation for
24hrs using Staphylococcal enterotoxin B. Cell-free fractions
from the activated and control cells were fractionated by twodimensional chromatography in the liquid and intact phase.
To improve the sensitivity of detection of protein signatures,
the secreted components were also subjected to a
fractionation strategy using IgY antibodies to deplete the
most abundant proteins in human serum and then analyzed
by two-dimensional liquid chromatography. Intact proteins
were separated by their isoelectric points in the first
dimension and further separated by hydrophobicity on a
second-dimension. The net result was the generation of highresolution protein profile of the complex mixture. Qualitative
and quantitative differences in protein profiles in activated
and non-activated cell free fractions could be easily identified
using powerful software. The use of the IgY fractionation
technique to deplete the abundant proteins in serum
containing growth medium enhanced the sensitivity of the
differential analysis. The gel- free and intact nature of the
fractions of interest allows for further interrogation and
identification of the differentially expressed proteins to
elucidate an activation signature in supernatants. Thus the
combination of proteomic techniques enables a more refined
and targeted profiling and analysis of complex events
associated with an immune response
ProteomeLab™ PF 2D Platform
Workflow
EXPERIMENTAL PROTOCOL
Increasing Hydrophobicity
ABSTRACT
Contact: [email protected], 305-380-2574
Proteins of interest that are differentially expressed in activated
supernatants are easily fractionated from complex mixtures in a
gel-free and intact state using the above described 2-dimensional
proteomic techniques enabling the identification of biomarkers of
activation
The proteins of interest can be further identified using other
techniques such as MS, peptide mapping, western blots, ELISAs
etc
Further integration of genomics, proteomics and cytomics
techniques can enable a holistic interrogation of biomarkers of
cellular activation