Proteomics Area

Reverse-Phase Protein microArrays (RPPA)

RPPA Laboratory Reverse-Phase Protein microArrays (RPPA) is a technological platform for antibody-based (phospho-)proteomic analysis. RPPA takes advantage of signal amplification protocols used in clinical diagnostic tests, to measure hundreds of analytes simultaneously on hundreds of biological samples, with elevated sensitivity and starting from as low as 25 micrograms of protein extract1)2)3). RPPA has been designed to study signal transduction pathways by relative quantification of proteins as well as their post-translational modifications. The type of samples that are suitable for such an analytical platform range from tissue and in vitro cultured cells to biological fluids such as urine, serum, saliva or cerebrospinal fluid. Therefore, RPPA is a multi-purpose, targeted analytical platform with a large spectrum of applicability4)5)6)7).

Over the past 5 years, the RPPA unit at ISS has gained a specific expertise in oncology. In particular, the platform has been used to:

  • study the mechanism of action (MoA) of small molecule kinase inhibitors;
  • discover biomarkers of drug sensitivity in cancer stem cells, based on pathway activation profiling.

Another innovative field of application for the RPPA technology is drug repurposing, whereby the effects of approved or orphan drugs on signal transduction networks may be studied on diverse model system to uncover novel, potentially translatable molecular effects.

The advanced equipment of the RPPA unit comprises:

  • a laser-capture microdissector with infrared laser, to cut user-selected portions of tissue from frozen tissue sections and minimize photo-damage of (phospho-)proteins;
  • a robotic, contact pin arrayer that allows micro-deposition of hundreds of samples on nitrocellulose-coated slides;
  • an autostainer, to allow automated immunostaining and signal amplification;
  • a laser scanner to digitalize the fluorescent signal from spotted samples on the slide surface;
  • a dedicated image analysis software for automated detection, quantification and normalization of signal.

Such a pipeline of instruments makes the RPPA unit at ISS an unparalleled laboratory implementation in Italy.

Moreover, the RPPA facility hosts a bank of > 400 antibodies directed against proteins, phosho-proteins and other post-translational modifications (Table 1) The number of antibodies undergoing validation for RPPA use is increasing, based on the requirement of novel project collaborations.

The RPPA unit at ISS has diverse internal collaborations as well as other national collaborators such as the National Cancer Institute in Milan and in Rome, the University of Rome “Tor Vergata”, the “Catholic University of Rome” and the University of Palermo.

The RPPA unit at ISS may provide technical and scientific support in experimental design, samples preparation and data analysis.

To ask for the service:

  1. preliminary contact the RPPA unit staff;
  2. fill out the online form 'Service Request' briefly illustrating the scientific problem, type and number of samples etc.;
  3. plan a meeting with the unit staff to discuss methods of analysis, costs, timing etc.

Contact: Michele Signore
+39 06 4990 4453
Where we are: GB-BS, room 7
1) Mueller C, Liotta LA, Espina V. Reverse phase protein microarrays advance to use in clinical trials. Mol Oncol. 2010 Dec;4(6):461-81. doi: 10.1016/j.molonc.2010.09.003. Epub 2010 Oct 16.
3) Gallagher RI, Espina V. Reverse phase protein arrays: mapping the path towards personalized medicine. Mol Diagn Ther. 2014 Dec;18(6):619-30. doi: 10.1007/s40291-014-0122-3.
4) Locard-Paulet M et al. Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration. Sci Signal. 2016 Feb 9;9(414):ra15. doi: 10.1126/scisignal.aac5820.
6) Gujral TS et al. Profiling phospho-signaling networks in breast cancer using reverse-phase protein arrays. Oncogene. 2013 Jul 18;32(29):3470-6. doi: 10.1038/onc.2012.378. Epub 2012 Sep 3.
7) Akbani R et al. A pan-cancer proteomic perspective on The Cancer Genome Atlas. Nat Commun. 2014 May 29;5:3887. doi: 10.1038/ncomms4887.