Reverse phase protein arrays (RPPA) have previously been used to provide, quantitative analysis of pathway responses at the post-translational level across multiple biological samples including clinical studies [1-4]. Tangible benefits of using a RPPA approach over alternative genomic or mass spectrometric proteomic methods include:
- Sample numbers are not limited by reagent costs or instrument throughput enabling robust analysis across clinical cohorts and preclinical temporal and dose-response studies.
- Precise and sensitive quantification of multiple pathway responses at a post-translational level including ratiometric analysis of drugable pathways that can be mapped directly to drug-target hypotheses including rationale combinations.
- Detection of low-abundant protein markers including signal transduction mediators in small sample material.
- Application of validated antibody based detection reagents that can be readily adapted to single or 2-3 plex diagnostic based assays.
The RPPA platform that we have invested in is the ZeptoMARK reverse protein microarray system provided by Zeptosens, a subsidiary of Bayer Technology Services GmbH. The ZeptoMARK platform uses proprietary planar waveguide technology to provide industry leading sensitivity and precise quantification of changes in protein levels and posttranslational modifications. Zeptosens have validated a portfolio of over 300 antibodies that can be used to simultaneously profile pathway response across multiple samples. Pathways covered include key PI3K, RAS-MAPK, Src/FAK, Rb/cell-cycle and TGF signalling axes and multiple DNA repair, cell-cycle, apoptosis mechanisms and epigenetic histone modifications. This is not an exhaustive list and can be adapted for other pathways. The array format of the ZeptoCHIP enables unlimited multiplexing and simple incorporation and validation of further antibody reagents as required. A published list of validated antibody reagents can be found on the Zeptosens website:
In addition, as members of a global RPPA network we are exploiting the expanding repertoire of specific antibody reagents by constantly adding more antibodies from ongoing validation studies to the probe lists, thereby increasing coverage of post-translational pathway analysis.
We believe that the Zeptosens detection system represents the most advanced and sensitive RPPA platform available and hence is our system of choice for routine pathway analysis at the post-translational level. The ZeptoMARK platform (installed within the Edinburgh Cancer Research Centre since March 2011) represents a fully integrated platform from automated sample handling to data analysis. The Edinburgh ZeptoMARK RPPA platform incorporates the following features:
- Automated sample handling and non-contact spotting of complex lysates under humidified and chilled environmental control provides high reproducibility of quantitative post-translational analysis.
- Chip and antibody assay barcode tracking integrated with a dedicated database and data visualization software (ZeptoVIEW 3.1) ensures robust data analysis and a seamless link between clinical or preclinical samples with antibody reagents and data analysis.
- Close interaction with the Edinburgh Experimental Cancer Medicine Centre, application of a cryo-controlled beadmill tissue homogenizer (FastPrep-24) and a dedicated post funded by the Edinburgh Cancer Research Centre to derive assured procedures for sample handling and antibody validation places us in an excellent position for applying the ZeptoMARK platform to translational studies.
Key application of our Zeptosens RPPMA platform include:
- Lead/candidate drug mechanism-of-action profiling – Quantifying on and off-target activities at the post-translational level.
- Biomarker discovery -in clinical and preclinical studies linked to dose/phenotypic outcome/context.
- Predictive pharmacodynamics-monitoring organ specific pathway effects of a compound in vivo.
- Drug combinations -define rational hypothesis and validate synergistic pathway activity. Assess impact of drug-drug interactions upon broad pharmacodynamic profiles of drug mechanism.
- Boyd, Z.S. et al. (2008) Proteomic analysis of breast cancer molecular subtypes and biomarkers of response to targeted kinase inhibitors using reverse-phase protein microarrays. Mol Cancer Ther 7 (12), 3695-3706
- Weissenstein, U. et al. (2006) Protein chip based miniaturized assay for the simultaneous quantitative monitoring of cancer biomarkers in tissue extracts. Proteomics 6 (5), 1427-1436
- Pawlak, M. et al. (2002) Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis. Proteomics 2 (4), 383-393
- Voshol, H. et al. (2009) Antibody-based proteomics: analysis of signaling networks using reverse protein arrays. FEBS J 276 (23), 6871-6879
- Internal Link: quantitative high resolution in vivo cancer imaging methodology
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