Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.
Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.
The clinical implementation of the targeted tyrosine kinase inhibitors (TKI) has transformed cancer treatment by providing physicians with effective tools to target the specific proteins that drive neoplastic transformation. These compounds inhibit or block the phosphorylation of proteins targeted by tyrosine kinases1,2. TKIs were developed in part because genetic alterations in various key signaling genes are sufficient to drive cancer initiation and progression [e.g.,epidermal growth factor receptor (EGFR), proto-oncogene tyrosine-protein kinase Src (SRC), BCR-ABL, and human epidermal growth factor receptor 2 (HER2)]3,4. The impact of TKIs on the cell cycle5 and the molecular signaling pathways6 represents a transformation from the untargeted to the molecularly guided cancer treatment. The key advantage of TKIs versus chemotherapy is the increased response rates and the lower risk of toxicity to healthy cells7. As a result, there has been increasing attention on the research and development of novel TKIs.
Access to the genomic sequencing results started with the Human Genome Project8,9,10 and continues today with various next-generation (NextGen) cancer sequencing efforts [e.g., The Cancer Genome Atlas (TCGA)11,12]. This has inspired many experimental methodologies that provide simultaneous information on thousands of genes and/or provide unbiased snapshots of genes or proteins modulated by biological perturbations13. Since the regulation of the cellular function occurs at multiple levels, from the transcription of genes to the post-translational modification of proteins and their activity, a complete understanding of the events controlling the cellular function will ultimately require an integration of data from various biological readouts. The ability to monitor the messenger RNA (mRNA) levels of thousands of genes with a single-cell gene resolution has increased the ability to make inferences about the gene function and interactions on a whole-genome scale. However, the interpretation of gene expression arrays will always be inherently incomplete without the integration of other levels of regulation: namely, the protein expression levels, the protein modification states, and the protein post-translational modifications (phosphorylation, ubiquitylation, methylation, etc.). Here, we describe the utility of antibody arrays as means to interrogate post-translational modifications of important signaling components as a function of various conditions in a single experiment14,15,16.
Phospho-antibody arrays can be employed to distinguish and analyze changes in the signal transduction pathways16. These can arise from a genetic modification or treatments of cell lines with kinase inhibitors, chemotherapeutics, stress caused by glucose deprivation, hypoxia, or serum starvation. Of note, drug resistance or a specific gene up- or downregulation can also cause changes in the signal transduction pathways17.
Drug resistance, for example, can arise from mutations of the drug target to avoid sensitivity. In lung cancer, known EGFR mutations render the cancer insusceptible to certain TKIs, but more susceptible to others. Alternative signaling pathways can be activated upon mutation17. As a broader application for the identification of the signal transduction pathways involved in resistance and hypoxia, etc., phospho-antibody arrays provide more insight into, and consequently understanding of, the mechanisms involved.
Technologies that permit an assessment of protein modifications represent an important component of systems biology because they often serve a regulatory function, such as modulating the activity of an enzyme or the physical interactions between proteins. The importance of post-translational modifications is illustrated by the role of protein phosphorylation in nearly all extracellular-triggered signal transduction pathways18. Traditionally, the identification of kinases or of the phosphorylation status of proteins could also be determined by Western blot analysis, especially if the researcher is interested in only 1–5 targets. However, Western blots are very selective and can be biased toward prior knowledge and might miss important targets as a result. Antibody array(s) provide a medium-throughput readout of multiple targets by embedding various capture antibodies [pan-specific phosphorylated tyrosine(s), anti-ubiquitin, etc.] on a solid matrix (e.g., glass or nitrocellulose). Secondary antibodies provide information on specific proteins in a sandwich-based ELISA format (Figure 1). This assay becomes more powerful and pertinent as more targets are of interest or the prior knowledge is restricted15. The phospho-arrays are more broadly employable as they can compare phosphorylation as well as general amounts of protein of a wider variety of targets in one experiment and provide a significantly improved quantification over mass spectrometry. This technique is not applicable for the identification of new or previously unknown phosphorylation sites.
Large-scale mass spectrometry-based proteomics could be employed to identify specific phosphorylation sites of proteins19. Although this technique can enumerate thousands of post-translational events, it requires expensive instrumentation, dedicated experimental pipelines, and a computational expertise that are beyond the reach of most researchers.
Antibody arrays provide a simultaneous readout on various protein readouts16. These may be changes in protein ubiquitylation (ubiquitin array) or phosphorylation. The key advantage of this array technology is that it provides important feedback on the biological state of various biological pathways associated with important cell parameters (protein of 53 kDa, p53, receptor tyrosine kinases, and intracellular pathways) simultaneously. In addition, it is possible to combine various array types to increase the penetration of an assay (e.g., apoptosis and ubiquitin and phosphorylation). This ability to combine multiple arrays to assess various post-translational alterations in several samples simultaneously in a time- and cost-effective manner that does not require specific instrumentation or expertise is of a significant advantage in the case of antibody arrays.
1. Protein Extraction
2. Human Phosphokinase Array
3. Data Analysis
To investigate the effect of TKI resistance on signal transduction pathways in cell lines, four samples were analyzed. One control sample (H3255r1) and 3 TKI-resistant cell lines (H3255r2-4) (Figure 2) were related to the spot antibody template (Figure 3). All 4 samples were prepared using this protocol. Six phosphoproteins with differential activity were chosen for the demonstration of the analysis of the antibody arrays (Figure 4). The heat map (Figure 5) provides a semi-quantitative readout on the changes in the protein phosphorylation of important signal transduction proteins. The validation of the array results can be completed by implementing orthogonal approaches, such as Western blotting.
Figure 1: Schematic of semi-quantitative antibody arrays. The antibody arrays are based on the sandwich immunoassay principle, where a collection of capture antibodies is embedded in either the glass or the nitrocellulose support. The cell lysates are incubated with the membrane and the arrays are subjected to secondary antibodies. A semi-quantitative readout may be obtained from chemiluminescent substrates or, for more quantitative information, fluorescent-based imaging. Any signals derived from film or TIF images may be processed by densitometry and a calculation of the fold-changes for each protein. The entire process takes about day. Please click here to view a larger version of this figure.
Figure 2: Example phospho-antibody array developed with the imager. The fluorescent spots are detected in duplicate (two spots per target). The samples and positive control spots show varying intensities. Shown here is the comparison of the TKI-sensitive H3255r1 and 3 resistant (H3255r2-4) sample arrays, each split in membrane A and B. Please click here to view a larger version of this figure.
Figure 3: Map of the array spots. The map links the spots to the antibody targets. The spots are labeled A – G vertically and 1 – 10 horizontally. The positive control (orange) is present on all membranes and usually found in the corners. The negative PBS control is highlighted in black while the sample spots are highlighted in yellow. For every array, a list of antibodies is also provided for identification. Please click here to view a larger version of this figure.
Figure 4: Data analysis example. (A) The intensity of the spots of interest, the positive control, and the background were identified on the map and measured for all samples. (B) The relative intensity (the spot intensity with the background intensity removed and normalized to a positive control intensity) is displayed in a bar chart for a few chosen candidates. Please click here to view a larger version of this figure.
Figure 5: Heat map of differential signaling in H3255 sensitive (r1) and resistant (r2-4) cell lines. A relative spot intensity was used to generate a heat map for a comparison of changes in CREB, P53, AKT, and STAT5 in all 4 samples. Any high levels are displayed in various shades of red while any lower levels are displayed in blue22. Please click here to view a larger version of this figure.
Approaches that combine many biological readouts are inherently more accurate representations of the cellular machinery in an experiment performed. The advent of phospho-antibody arrays enables a rapid characterization of the pattern of modifications which may be more informative than the modification status of any single protein. The general workflow for the application of phospho-antibody arrays is based on a modification in serine, threonine, or tyrosine. This example focused on characterizing the changes associated with the resistance to therapy in lung cancer. The main rationale for this application is that protein phosphorylation plays a central role in the signal transduction in many human cancers18, and this method will be transferable to other systems. A dysregulation of phosphorylation in cancer usually involves the hyperactivation of a tyrosine kinase, and consequently, the phosphorylated substrates (often including the auto-phosphorylated kinase itself) are present at levels higher than in the normal physiological settings and, accordingly, can make up a significant fraction of the phosphoproteins in a cancer cell. These higher levels of phosphorylation will make detection easier. Moreover, protein phosphorylation has a medical significance since aberrant tyrosine phosphorylation is a hallmark of many types of cancer23. In addition, since this technique is transferable to the investigation of other biological systems that utilize phosphorylation, many of the features described here could easily be adjusted to focus on other types of protein arrays (ubiquitin, apoptosis, etc.).
Phospho-antibody arrays are widely used for the identification of any signal pathways involved, either as an exploratory method or as verification of one particular pathway. Various companies make kits for both the phosphorylation status and for the overall levels of proteins. While the real-time polymerase chain reaction (PCR) analysis or microarrays are much more quantitative, they take only mRNA levels into consideration, and a translation as well as post-translational modifications cannot be addressed. Apart from phosphorylation, other post-translational modifications such as glycosylation or ubiquitylation can also be addressed by arrays24,25.
One of the important factors to consider before starting antibody arrays is to start with healthy and actively dividing cells. Hypoxia, oxidative stress, and inflammation may produce changes in the signal transduction pathways26 that can skew the results and render a misinterpretation if treated improperly. This stress can be attributed to the formulation of media or to plating too little or too many cells, or to exposing the cells to poorly regulated environments, or to treating the control versus the sample(s) slightly differently. We have also noted large differences in the passage number or the time in the culture post-thawing. The key to avoiding these artificially introduced variables is to keep everything between the samples as consistent as possible. Do not change the FBS percentage or the media volume, etc., during the experiment.
The data analysis should also be considered before starting an array experiment. A chemiluminescent array analysis is only semi-quantitative, and its dynamic range is approximately one order of magnitude, while a fluorescent approach increases the dynamic range to approximately two orders of magnitude. The scale on which arrays are performed depends on the specific biological question(s). It is possible to analyze one specific resistant cell line of interest for all changes in the post-translational modifications compared to the original cell line or to focus on one particular pathway for a wide variety of resistant cell lines. The scalability of this approach should be considered at the early planning stages. Furthermore, the results of an antibody array should always be confirmed by a secondary method on fresh samples and/or the same lysates. A western blot analysis can be employed to verify the changes to specific targets.
The authors have nothing to disclose.
We are grateful for the generous financial support of the Lawrence J. Ellison Institute of Transformative Medicine of USC (a gift to David Agus). We appreciate the support of Autumn Beemer and Lisa Flashner which lead to the generation and publication of this manuscript. We thank Laura Ng for her administrative support.
Odysee SA Imager | Li-Cor Biosciences | Fluorescent Imager | |
1.5 ml tube | Eppendorf | 22363212 | |
Cell Scraper | Falkon (Corning) | 353085 | |
Dulbeco's Phosphate buffered Saline (PBS) | Corning | 21-031-CV | wash buffer for protein extraction |
Tissue Culture dish 100mm | TRP | 93100 | |
ICC Insulin syringe U100 | Becton Dickinson | 329412 | 27G5/8, 1ml for needle treatment of protein samples |
Protein Profiler ARRAY | R&D | ARY003B | Human phospho MAPK array |
Protein Profiler ARRAY | R&D | ARY002B | Human phospho kinase array |
Centrifuge Eppendorf 5430R | Eppendorf | Table top centrifuge | |
Pierce BCA protein assay kit | Thermo Fisher | 23225 | |
SpectraMAX M2 | Molecular Devices | Absorbance reader for protein quantification | |
IRDye 800CW Streptavidin | Li-Cor Biosciences | 925-32230 | Streptavidin conjugate for fluorescent detection |
LabGard ES Class II, Type A2 biosafety cabinet | NuAire | NU-425-400 | Tissue culture hood |
TC20 automated cell counter | Bio-Rad | 1450102 | Cell counter |
Halt Protease & Phosphatase Inhibitor Cocktail (100X) | Thermo Fisher | 78446 | |
RIPA buffer | Sigma | R0278 | |
Sonic Dismembrator | Fisher Scientific | F60 | sonicator |
Rocking platform shaker | VWR | 10860-780 | |
ImageJ | NIH open source | https://imagej.net/Welcome | |
SAS Institutie JMP® 12.1.0 (64-bit) | Microsoft |