The Simple Western Rapidly Generates Quantitative Profiles of MAPK and PI3K Proteins in Clinical Specimens - Dr. Alice Fan, Stanford University

In this era of targeted therapeutics, there is a need for highly sensitive and quantitative methods to measure proteins in clinical specimens to define and predict of specific therapies for patients. However, the current gold standard in proteomic analysis, Western Immuno Blotting, has many manual steps, is insensitive and provides only semi-quantitative data. Here, we present the first report of a novel automated instrument “Sally^TM,” a nanovolume size-based protein separation platform used to quantify proteomic profiles of clinical specimens. Since the instrument automates all steps of proteomic analysis including sample loading, size-based protein separation, immunoprobing, washing, detection and data quantification, we are able to make up to 96 measurements in a single experiment, in a highly quantitative manner, minimizing errors due to manual variability. We were able to quantify proteins in either surgical specimens preserved at -80C in Optimum Cutting Temperature Compound (OCT) or fine needle aspirates flash-frozen at time of collection. Tissues were homogenized and lysed in a commercially available Bicine/CHAPS lysis buffer, denatured and loaded in duplicate in a 384-well plate at a final concentration of 0.2-2 mg/ml in each well. 40 nanoliters of lysate was used for each protein measurement. We measured 10 proteins (including AKT and ERK) from the MAPK signaling pathways, normalized to loading controls (β-actin or tubulin). It took 1 hour to prepare a run and 14 hours of unattended automated machine time to generate the analyzed data. Therefore this technology enables us to report highly quantified protein profiles to the clinical team in less than 24 hours of receiving a clinical sample. In the course of 1 week, we quantified ERK1 and ERK2 in over 100 clinical specimens and in a subset of 22, measured an additional panel of proteins including PI3K proteins [AKT1, AKT2, pan-AKT, GSK3b, S6]. The ability to make rapid and quantitative measurements will be useful to measure and predict responses to targeted therapeutics. The Sally platform is an extremely efficient, quantitative and high-throughput platform that can be used to rapidly generate proteomic profiles for clinical specimens for the development of novel diagnostic and predictive biomarkers.