Optimization of RPPA Protocols for FFPE Tissue: A Comparison of Protein Extraction, Protein Printing, and Total Protein Staining Methods

Abstract

Reverse Phase Protein Array (RPPA) is a technique that microprints proteins from a given sample onto a nitrocellulose coated slide, and utilizes specific antibodies to measure protein expression. These arrays can be constructed using proteins extracted from a variety of sample materials such as formalin-fixed paraffin-embedded (FFPE) tissue. However, there has been debate over the reproducibility of methods used to extract proteins and break formaldehyde cross-links of FFPE tissue for downstream analysis. Currently, the CAPMM lab constructs RPPAs using solid-pin printing, but are in the process of introducing a new inkjet style printing process in addition to a new laser scanning system. The new printing process deposits a smaller amount of total protein per spot on the microarrays, which can make high backgrounds problematic for data analysis. Additionally, the new laser scanning system is not compatible with the current total protein stain, and the high sample background that is frequently observed with FFPE extracted protein can be obstructive for total protein normalization and downstream analysis. These issues require modified protocols for protein extraction, sample printing, and total protein staining. In this experiment, RRPA arrays were constructed using protein from FFPE tissue collected via Laser Capture Microdissection (LCM) to compare the new printing process, extraction buffer, and new total protein stain with current protocols. The results of this experiment showed that the inkjet printing process produces smaller spots, thus reduces total protein printed. Overall, a decreased background was observed in Fast Green vs. SYPRO Ruby Red total protein staining. Finally, reduced background from TCEP vs. Qproteome® buffer extracted protein showed mixed results. These findings will help improve in-house modification of current protocols.