Dual-Color, Multiplex Analysis of Protein Microarrays for Precision Medicine

Abstract

Generating molecular information in a clinically relevant time frame is the first hurdle to truly integrating precision medicine in health care. Reverse phase protein microarrays are being utilized in clinical trials for quantifying posttranslationally modified signal transduction proteins and cellular signaling pathways, allowing direct comparison of the activation state of proteins from multiple specimens, or individual patient specimens, within the same array. This technology provides diagnostic and therapeutic information critical to precision medicine. To enhance accessibility of this technology, two hurdles must be overcome: data normalization and data acquisition. Herein we describe an unamplified, dual-color signal detection methodology for reverse phase protein microarrays that allows multiplex, within spot data normalization, reduces data acquisition time, simplifies automated spot detection, and provides a stable signal output. This method utilizes Quantum Nanocrystal fluorophore labels (Qdot) substituted for organic fluorophores coupled with an imager (ArrayCAM) that captures images of the microarray rather than sequentially scanning the array. Streamlining and standardizing the data analysis steps with ArrayCAM high-resolution, dual mode chromogenic/fluorescent array imaging overcomes the data acquisition hurdle. The spot location and analysis algorithm provides certain parameter settings that can be tailored to the particular microarray type (fluorescent vs. colorimetric), resulting in greater than 99 % spot location sensitivity. The described method demonstrates equivalent sensitivity for a non-amplified Qdot immunoassay when using automated vs. manual immunostaining procedures.

Proteomics appending a complementary dimension to precision oncotherapy

“…Accurate protein quantification relied on the identification of protein specimens in images acquired with high precision, followed by speckle analysis. Speckle analysis was capable of distinguishing speckles and measuring the pixel intensity of each speckle concerning the surrounding background [62] , [63] .…”

Pilot clinical trial and phenotypic analysis in chemotherapy-pretreated, metastatic triple-negative breast cancer patients treated with oral TAK-228 and TAK-117 (PIKTOR) to increase DNA damage repair deficiency followed by cisplatin and nab paclitaxel

“…Immunostaining was performed on a Dako Autostainer per manufacturer's instructions (CSA kit and Genpoint kit, Agilent) (29,31). Each slide was incubated with a single primary antibody at room temperature for 30 minutes (Supplemental Table 2), and antibody speci city was con rmed by Western blotting as previously described (32).…”