Let’s Get Cooking: Can an Instant Pot be used to improve protein extraction from clinical samples?

Abstract

Reverse Phase Protein Array (RPPA) is a protein identification technique that involves printing an array of proteins onto a nitrocellulose slide and using antibodies to detect and amplify the signal for specific proteins from target cells. Formalin-fixed paraffin-embedded (FFPE) tissue is the standard for preserving tissue used in clinical laboratory settings, including in the RPPA processes. However, during the formalin fixation process, formaldehyde cross-links are formed which can obscure antigen-antibody binding regions and interfere with subsequent downstream analysis. Currently, industry standard extraction and antigen retrieval processes occur at atmospheric pressure (~ 14.7 psi) and use the costly Q-Proteome® buffer which can show poor protein-to-background ratio on RPPAs, making downstream analysis challenging. Currently, the CAPMM lab is in the process of introducing a new RPPA printing process that utilizes inkjet style printing, which differs from the standard process of solid-pin printing. This new process requires a larger printing buffer volume, making low protein concentration outputs unviable. With this, we compared two buffers - TCEP [tris(2-carboxyethyl) phosphine] buffer, which in previous tests yielded lower background against Q-Proteome®. Additionally, both buffers underwent pressure and temperature tests: atm at 80℃, or placed in an Instant Pot®, at ~24-26psi at ~120℃, to potentially increase extraction efficiency and cross-link breakage. A RPPA was constructed and analyzed via an established fluorescence-based detection methodology to determine antigen binding retrieval and protein integrity. Total protein extraction decrease was observed with the TCEP buffer (p = 0.0195), whereas instant pot extraction consistently yielded higher concentrations (p = 0.0072). Decreased antigen binding was observed with instant pot extraction (p < 0.0002) in six out of seven antibodies. Significantly decreased protein counts were observed with TCEP extracted samples in three of the seven antibodies (p < 0.05) tested but showed no significant effect on the remaining four antibodies tested. Refining the TCEP recipe and extraction procedure may improve protein extraction and antigen binding proficiency and better prepare samples for inkjet printing required in the future.