Comprehensive Mapping of Protein Expression Profiles in Activated and Non-Activated Peripheral Blood Mononuclear Cells
Sybil D'Costa
Cell Development Analysis Center
Beckman Coulter, Inc.
Materials and Methods
Purification of PBMCs from Whole Blood
Heparinized peripheral blood was obtained from normal donors. Purified peripheral blood mononuclear cells were isolated using Ficoll-Paque* density gradient centrifugation.1 The cells were resuspended in freezing medium (90% fetal calf serum and 10% DMSO) at 107 cells/mL and frozen at -80°C.
Activation of PBMCs
Frozen PBMCs were thawed rapidly and washed once by resuspending the cells in RPMI with 10% fetal calf serum, glutamate, and penicillin/streptomycin, centrifuging the cells at 300 × g for 10 minutes, and further resuspending the cells in medium at a concentration of 107 cells/mL/15-mL conical tube. The activated cultures were treated with SEB at 2 µg/mL, anti-CD28 at 0.15 µg/mL, and incubated at 37°C for 6 hours, while the control cultures were left untreated in medium and also incubated at 37°C for 6 hours.
Preparation and Running of Samples for Analysis on the PF 2D System
At the end of the incubation period, cultures were centrifuged at 400 x g for 10 minutes, and the cell pellets were resuspended in 0.4 mL of 50 mM Tris (pH 7.8-8.2) and 1.6 mL of lysis buffer with vigorous shaking as per lysing of human cells protocol.2 The cell lysates were centrifuged at 20,000 x g for 60 minutes and the samples run on the 1st and 2nd dimension as described.3
Results
Activation of PBMCs
In order to analyze changes in protein profiles associated with an immune response, the superantigen SEB and co-stimulator anti-CD28 were chosen to activate PBMCs. Superantigens are unique antigens that are able to activate 1–20% of the total T cell population by binding to the outer face of specific T cell receptor (TCR) subsets on T cells and crosslinking them to MHC Class II molecules (see Figure 1).4 Purified PBMCs were activated with SEB and anti-CD28 for 6 hours at 37°C or were left untreated. Control and activated cell cultures were harvested by centrifugation. The cell pellets were then lysed in lysis buffer as described in the Methods section and run on the PF 2D platform.
Figure 1. Representative schematic of the interaction of staphylococcal
enterotoxin B (SEB) with T cell and antigen presenting cell (APC). SEB
recognizes specific VB segments of the T cell receptor (TCR) and specific
regions on the MHC molecule outside the peptide-binding groove thus
enabling contact between the T cell and APC and subsequent activation of
the T cell.
Analysis of Control and Activated Protein Profiles Using the ProteomeLab Mapping and Visualization Software
Using the ProteomeLab visualization software, the 2D pI/hydrophobicity maps for control and activated cultures were examined. Figures 2A and 3A are the derived 2D maps for the control and activated cultures, respectively. Lanes 2–17 represent all proteins eluting within specific pI ranges, with lane 2 containing all the proteins having pI values < 4, and lanes 3–17 representing pI ranges of 4–8.5. The 2nd-dimension, reversed-phase analysis furnishes hydrophobicity profiles of each pI fraction. The "zoom-in" feature of the software enables detailed analysis of small sections of the 2D pI/hydrophobicity map and allows the rapid visualization of proteins expressed at low concentrations.
Figure 2. Derived map stratifying proteins in control PBMC cultures
with pI ranges from 4.0-8.5. PBMCs left untreated at 37º C for 6 hours
were centrifuged and the cell pellets were treated to the lysis protocol.
The lysates thus obtained were run on the 1st- and 2nd-dimension
separation protocols, and the protein profiles were mapped using the
visualization software. Figure 2B is a zoom in of the 2D pI/hydrophobicity
map of control cultures to lanes 7-9 (pI 5.2-6.1) and running at retention
times 14-17 minutes.
Figure 3. Derived map stratifying proteins in control PBMC cultures
with pI ranges from 4.0-8.5. PBMCs activated with SEB and anti-CD28 were
incubated at 37º C for 6 hours, centrifuged, and the cell pellets were
treated to the lysis protocol. The lysates thus obtained were run on the
1st- and 2nd-dimension separation protocols, and the protein profiles were
mapped using the visualization software. Figure 3B is a zoom-in of the 2D
pI/hydrophobicity map of activated cultures to lanes 7-9 (pI 5.2-6.1) and
running at retention times 14-17 minutes.
Analysis of Control and Activated Protein Profiles Using the Differential Display Capabilities of the Protein Mapping Software
The differential display software enables the comparison of protein profiles between samples and allows for the effortless visualization of proteins that are differentially expressed between the samples. Figure 4 is representative of the differential mapping capability, wherein Figure 4A is a 2D pI/hydrophobicity map of the proteins in the control culture, and Figure 4C is a 2D pI/hydrophobicity map of the proteins in the activated culture, with the proteins having pIs ranging from 4.0–8.5 and retention times ranging between 7–20 minutes on the 2nd dimension run. Figure 4B is the differential map enabling the identification of proteins differentially expressed in the control and the activated cultures. Thus, the differential display allows for quick identification of proteins of interest.
Figure 4. 2D pI/hydrophobicity map (pI 4.0-8.5, retention times 7-20
minutes) of control and activated PBMC cultures. PBMCs either left
untreated or activated with SEB and anti-CD28 were incubated at 37º C for
6 hours, centrifuged, and the cell pellets treated to the lysis protocol.
The lysates thus obtained were run on 1st- and 2nd-dimension separation
protocols, and the protein profiles were mapped. Figures 4A and 4C are
the 2D pI/hydrophobicity maps of the control and activated cultures, respectively. Figure 4B is a differential protein profile expression
between control and activated cultures.
References
- Tripodi, D., et al. "Separation of peripheral leukocytes by Ficoll density gradient centrifugation." Transplantation 11 (5), 487-488 (1971).
- ProteomeLab PF 2D Kit Use Protocol, P/N 390970aa.
- Simonian, M. H., Betgovargez, E. "Proteome Analysis of Human Plasma with the ProteomeLab PF 2D System." Beckman Coulter Application Information Bulletin A-1963A.
- Krakauer, T. "Immune responses to staphylococcal superantigens." Immunol. Res. 20, 163-173 (1999).
* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.
