Reverse Phase Protein Array (RPPA) Core Facility
Yiling Lu, Rehan Akbani
Director, Co-Director
- Research Resources
- Core Facilities and Services
- Functional Proteomics RPPA Core Facility
Overview
The Reverse Phase Protein Array (RPPA) Core provides investigators with a powerful, high‐throughput, quantitative, cost‐effective technology for functional proteomics studies. Furthermore, we provide centralized, standardized and quality-controlled services to investigators not only throughout MD Anderson, but around the world, as well as to several national consortia, including TCGA, CCLE and ICBP.
Advantages of RPPA and Functional Proteomics
RPPA
The core has defined a set of standard lysates to be used in each array for quality control (QC) of data generation and analysis. A set of algorithm and custom software applications has been developed to improve quantification and quality control.
- Cost effective: A high throughput approach concurrently tests and quantifies over 1000 samples on a signal slide.
- Sensitive: Applicable to very small sample sizes (ng of protein lysates, detecting attomoles of aspecific protein), less than 10 cell equivalents. It detects and quantifies hundreds of different proteins at expression levels and modification status in 40 ug of cell lysates.
- Quantitative: Recombinant protein/phosphopeptides enable absolute quantification of protein expression and modification levels.
- Simple: Does not require direct labeling of the lysate sample.
Functional Proteomics
Studies of complex diseases such as cancer have shown that genetic alterations do not account for all of the causes of the disease. Changes in protein levels and structure have also been shown to play critical roles in tumor development and progression, which are not reflected by genetic changes. In cancers, several genetic and epigenetic changes are often required for development of the disease. Studying large-scale epigenetic changes such as protein phosphorylation or cleavage will greatly aid in understanding the causes and determining effective treatment of cancers and other complex diseases.
Our Unique Platform
MD Anderson's RPPA Core is a valuable resource to the scientific community due to its unique and continuous validation and quality control of all aspects of its RPPA Pipeline, including the below:
- Serial dilutions of each sample to capture the linear antibody/antigen reaction for accurate data analysis
- An ongoing and highly stringent antibody validation process to select and maintain only the highest quality antibodies used for RPPA analysis
- An “anchor” of 48 unique cell lysates printed on every slide that serve as controls to develop Replicate-Based-Normalization used for quality control for data generation and analysis and RPPA data merging across different slides
- A series of rigorous quality control processes developed, implemented, and optimized for our RPPA data analysis to provide reliable data to customers: algorithms of spatial correction, quality control of antibody probing, protein loading correction, replicate-based-normalization, and quality of antibody batches
- An automated program for RPPA Pipeline processes implemented to reduce manual labor, human error, and customer turnaround
- A set of algorithm and custom software applications developed to improve quantification and quality control
Method
RPPA represents an antibody‐based functional proteomic analysis for both tumor tissue and cultured cells. RPPA characterizes the basal protein expression and modification levels, growth factor‐ or ligand‐induced effects, and time‐resolved responses appropriate for systems biology analysis. It provides information to integrate the consequence of genetic aberrations in cancer, to validate therapeutic targets, to demonstrate on‐ and off‐target activity of drugs, and to evaluate drug pharmacodynamics.
Core Grant Citations
The RPPA Core is supported by NCI Grant # CA16672 and Dr. Yiling Lu’s NIH R50 Grant # R50CA221675: Functional Proteomics by Reverse Phase Protein Array in Cancer. Please cite these grants in the acknowledgment section if your publications use data generated by the RPPA Core. Two copies of the publication acknowledging these grants should also be submitted to the RPPA Core at Unit 1058. Thank you!