Tyrosine phosphorylation profiling via in situproximity ligation assay
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Tyrosine phosphorylation (pTyr) is an important cancer relevant posttranslational modification since it regulates protein activity and cellular localization. By controlling cell growth and differentiation it plays an important role in tumor development. This paper describes a novel approach for detection and visualization of a panel of pTyr proteins in tumors using in situ proximity ligation assay.
K562 leukemia cells were treated with tyrosine kinase and/or phosphatase inhibitors to induce differences in pTyr levels and mimic cells with different malignant properties. Cells were then probed with one antibody against the pTyr modification and another probe against the detected protein, resulting in a detectable fluorescent signal once the probes were in proximity.
Total and protein specific pTyr levels on ABL, SHC, ERK2 and PI3K proteins were detected and samples of control and treated cells were distinguished at the pTyr level using this novel approach. Promising results were also detected for formalin fixed and paraffin embedded cells in the micro array format.
This application of in situ proximity ligation assay is valuable in order to study the pTyr modification of a panel of proteins in large data sets to validate mass spectrometric data and to be combined with tissue microarrays. The approach offers new opportunities to reveal the pTyr signatures in cells of different malignant properties that can be used as biomarker of disease in the future.
KeywordsCancer biomarkers Protein signaling Protein tyrosine phosphorylation in situ proximity ligation assay (in situ PLA)
Proximity ligation assay
Post translational modification amplification
Tissue micro array
Tyrosine phosphorylation (pTyr) of proteins is an important posttranslational modification (PTM) that regulates many essential cellular functions . The modification is often involved in development and progression of cancer [2, 3]. The identification of this modification is therefore important in order to understand systems biology. PTMs such as phosphorylations of proteins are commonly identified by tandem mass spectrometric (MS) methods after phosphopeptide enrichment via immunoaffinity or chemical affinity methods [4, 5, 6]. The MS method is an excellent approach to reveal the PTMs and to map specific amino acids that carry the modifications on several different proteins. There is, however, a need for complementary tools for i) confirming the findings, ii) for measuring the abundance of a certain PTM in large collections of small amounts of clinical tumor materials and iii) to reveal PTMs on low-abundant proteins in complex matrices where increased specificity is required. For this purpose, a quantitative approach using MS detection is not the method of choice. Instead a fast method that can handle many samples at the same time e.g. using western blotting, ELISAs or as presented in this study, proximity ligation assays (PLA) with PTM specific antibodies is advantageous.
The in situ PLA that was developed in 2006 is now an established technique for detection of individual proteins, protein-protein interactions  as well as PTMs in cell lines and tissue sections [8, 9, 10, 11]. Briefly, when DNA oligonucleotides coupled to antibodies against different epitopes of proteins are in proximity, they will hybridize to a couple of DNA oligonucleotides and template a subsequent enzymatic ligation to form a circularized DNA molecule. The newly formed circularized DNA molecule will then be amplified using rolling circle amplification (RCA). The technology has unique specificity due to the dual recognition and extreme sensitivity in the pM-fM range in comparison to the sensitivity of shotgun MS in the μM-nM range . In addition, it provides visualization of the cellular location of studied proteins.
All chemicals were from Sigma Aldrich (St. Louis, MO, USA) if not otherwise stated. The pTyr monoclonal antibody 4G10 was from Millipore (Billerica, MA, USA) and the PYKD1 monoclonal antibody was purchased from Sloan-Kettering Institute for Cancer Research (New York, NY, USA). The protein specific primary polyclonal antibodies used in this study were anti-c-ABL (sc-131) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-SHC (610082) from BD Transduction Laboratories (Franklin Lakes, NJ, USA), anti-ERK2 (06–333) from Millipore (Billerica, MA, USA), anti-PI3 kinase p110 β (#3011) and anti-PI3 kinase p85 (#4292) from Cell Signaling (Boston, MA, USA).
The leukemia K562 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Life Technologies, Stockholm, Sweden). Before harvesting the cells, half of the cell culture was treated with 50 μM Imatinib (imatinib mesylate, Glivec or Gleevec, Novartis, Basel, Switzerland) for 1 h or 100 μM pervanadate for 30 min. Pervanadate was prepared from ortovanadate reacted with 0.05% hydrogen peroxide for 5 min. Excess of hydrogen peroxide was removed by incubating the mixture at room temperature (RT) with catalase to a final concentration of 200 μg/mL.
Cells were adjusted to a concentration of approximately 0.5×106 cells/mL by diluting with cell culture medium. One mL cell cultures were then pelleted by centrifugation, and washed once with PBS and twice with Ca, Mg and NaHCO3 free PBS (Life Technologies, Stockholm, Sweden). Both PBS solutions were supplemented with 1 mM Na3VO4. After the final wash, cells were dissolved in 1 mL Ca, Mg and NaHCO3 free PBS and kept at 4°C until use. Cells were spotted on glass slides (Superfrost, Plus Gold, Thermo Scientific, Braunschweig, Germany) with Shandon filter cards (Thermo Electron Corporation, Runcorn Chesire, United Kindom) using the cytospin technology (Cytospin 2, Shandon, Thermo Fisher Scientific, Waltham, MA, USA). Briefly, 150 μL of cell solution was added per glass holder (giving approximately 75,000 cells per glass), and spun at 500 rpm for 5 min. Cells were then fixed in 70% ice-cold ethanol for 30–60 minutes, thereafter the slides were left to dry. Prepared slides were stored at -20°C until used.
In situproximity ligation assay experiments
The in situ PLA experiments were performed using reagents and instructions found in commercially available kits from Sigma-Aldrich; Duolink® In Situ PLA® Probe Anti-Rabbit MINUS/PLUS (DUO92005/DUO92002), Duolink® In Situ PLA® Probe Anti-Mouse MINUS/PLUS (DUO92004/DUO92001) and Duolink® In Situ Detection Reagents Orange (DUO92007). The fixed samples on the slides were encircled with a hydrophobic pen (ImmEdge Hydrophobic barrier pen, Vector Laboratories, art no. H-4000) and rehydrated in 1 × TBS for 15 min. Forty μL of blocking solution was added to each sample and the slides were incubated in a humidity chamber for 1 h at 37°C. Primary antibodies were diluted to final concentrations of 1 μg/mL, except for anti-SHC antibody that was used at 0.1 μg/mL concentration. When using only the 4G10 antibody in in situ PLA, the range of 0.1-1 μg/mL was used. Blocking solution was removed and 40 μL of primary antibody mix was added to corresponding sample, and slides were incubated in a humidity chamber overnight (approximately 22 h) at 4°C. Secondary probes were diluted to final concentrations of 1:5 in antibody diluent (supplied in the kit). Primary antibody solution was removed and the slides were washed in 1 × TBS 0.05% Tween 20 (TBS-T) for 2 × 5 min with gentle agitation (shaker set at ~60 rpm) (hereby referred to as the washing procedure) before 40 μL of the secondary probes mix was added to each sample. The slides were incubated in a humidity chamber for 1 h at 37°C, washed (see washing procedure), and thereafter 40 μL ligation solution was added to each sample. The slides were then incubated in a humidity chamber for 30 min at 37°C, ligation solution was removed, slides were washed (see washing procedure), and 40 μL amplification solution (MilliQ H2O, Amplification/detection mix, polymerase) was added to each sample. The slides were incubated in a humidity chamber for 100 min at 37°C. Slides were then washed briefly in 1× TBS before 40 μL of 100 µg/mL (in 1 × TBS) of Hoechst dye (Life Technologies, art. No. H1399) was added to each sample to stain cell nuclei for fluorescence microscopy. Samples were incubated in dark for 1 h at RT and then washed in dark in MQ water over night. Before detection, 10 μL of mounting media (Slow Fade Gold Antifade Reagent, Life Technologies, art. No. S36936) was added to each sample and covered with a cover slip. Edges of the cover slip were sealed with nail polish. Slides were stored at 4°C.
Microscopy and data handling
The results were viewed with an epifluorescence microscope (Axioplan II, Zeiss, Germany) and stacked TIFF files were obtained using Axiovision 4.8 (Zeiss, Germany), with a maximum slice distance of 0.5 μm in order to image every PLA signal. Since the number of signals per cell can vary within a sample mainly due to that cells are in different stages of cell cycle, but also because of local differences in reagent concentration, cell confluences and drying, a minimum of at least five images were taken for each sample at various sites on the slide. Also, in order to account for the cell-to-cell variation in the number of signals per cell, and to obtain significant quantifications, a total numbers of 100–200 cells were imaged from each sample. The images were analyzed in Duolink image tool (Sigma-Aldrich, Germany), the drawing tool was used to exclude background signals on the slide and partial cells. The quantifications were exported to Excel and the numbers of nuclei were controlled manually for each image within the drawn area. Data were compiled along with standard deviations. Statistical analyses were performed in GraphPad Prism (GraphPad software Inc., La Jolla, CA, USA). Signals for each experiment were plotted in a diagram with marked average and bars corresponding to 95% confidence intervals to visualize the significance level of experiments. Two outliers were removed (in two different experiments) after statistic outlier test.
Formalin fixed paraffin embedded samples
Forty-six in vitro cultured cell lines (among others the K562 and U937 cell lines) representing different cell types were fixed in formalin after harvest. The fixed cells were then mixed with melted agarose, in order to create a block of dispersed single cells in a 3D matrix. The resulting cell gels were subjected to standard histoprocessing, paraffin embedding, and finally assembled in a cell microarray format .
Western blot were used to evaluate the performance of primary antibodies used, and is further described in Additional file 1.
Results and discussion
The pTyr modification is a disease relevant PTM since its dysregulation can lead to uncontrolled growth, which is a hallmark of cancer. It is therefore of importance to evaluate novel approaches that can be used for its detection. Compared with MS detection, in situ PLA-based detection of pTyr proteins can be performed using much less material, and there is no need for extensive sample preparation before measurement. Therefore, larger sample sets can be screened for the signature of the pTyr modification and pTyr sites discovered by MS detection can also be verified since the two methods have different selectivity mechanisms and should be considered complementary. Furthermore, the in situ PLA can be applied on intact cells and tissue sections, also enabling determination of localization and a tool to follow the dynamics in the localization of the pTyr proteins in the cells. In this study we present a general approach for analysis of the overall pTyr pattern form all pTyr proteins as well as an established protocol to detect the pTyr levels for a panel of specific cancer relevant proteins. In the in situ PLA used in this study one antibody against the target protein was combined with an anti-pTyr antibody (Figure 1). An average value of the pTyr level of the protein was then detected, but site specific information was not available. The usage of a general pTyr antibody instead of expensive site-and-protein-specific pTyr antibodies, leads to a reduced cost and therefore wider applicability of this method. The model system for testing the PLA approach for pTyr proteins was the K562 leukemia cell line. This cell line expresses the BCR-ABL fusion protein, and the ABL kinase is constitutively active and produces an overall relatively high pTyr level. Since the ABL kinase can be controlled via imatinib treatment – a tyrosine kinase inhibitor (TKI) that decreases the pTyr levels – a measurable difference is introduced. Further, since the K562 cell line responds to pervanadate treatment – a general tyrosine phosphatase inhibitor (TPI) – an induced increase in pTyr levels can be established. Previously, we described characterization of more than 200 pTyr proteins in the K562 cell line using MS detection  from which a subset was selected for this study to be tested by in situ PLA.
Development of new approaches to study the pTyr modifications is of great importance and is likely to improve the understanding of cellular signaling and thereby disease related events. In the future, pTyr signature patterns are likely to enable classification of tumors and to reveal perturbed pathways. As described in this paper, the in situ PLA method using one antibody against the phosphorylation and another against the specific protein is a successful approach. In this way perturbations in pTyr levels can be detected with cellular resolution in small sample amounts.
This research was performed with support from Swedish Cancer Society (SBL), P.O Zetterling Foundation (SBL), Åke Wiberg Foundation (SBL) and Kjell and Märta Beijer foundation (UP), GastricGlycoExplorer (MKM). Lena Claesson-Welsh is acknowledged for providing test aliquots of antibodies. The in situ PLA experiments were performed at PLA proteomics facility at Swedish Science for Life Laboratory.
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- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/435/prepub
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