Analysis of Individual Signaling Complexes by mMAPS, a Flow‐Proteometric System

Abstract

Signal transduction is essential for maintaining cells’ normal physiological functions, and deregulation of signaling can lead to diseases such as diabetes and cancers. Some of the major players in signal delivery are molecular complexes composed of proteins and nucleic acids. This unit describes a technique called microchannel for Multi-parameter Analysis of Proteins in a Single-complex or mMAPS for analyzing and quantifying target signaling complexes individually. mMAPS is a flow-proteometric-based system that allows detection of individual proteins or complexes flowing through a microfluidic channel. Specific target proteins and nucleic acids labeled by fluorescent tags are harvested from tissue samples or cultured cells for analysis by the mMAPS system. Overall, mMAPS enables both detection of multiple components within a single complex and direct quantification of different populations of molecular complexes in one setting in a short timeframe, requiring very low sample input.

Structural and Functional Impacts of ER Coactivator Sequential Recruitment

“…In an attempt to confirm that the dissociation of one SRC-3 from the ER complex upon CARM1 binding also occurs in cells, we utilized an advanced Flow Proteometric system (Chou et al, 2014; Chou et al, 2016) to analyze the stoichiometry of SRC-3 and ER within a single ER complex in the absence or presence of CARM1. HeLa cells were transiently transfected with ERα with or without CARM1 co-transfection and stimulated with estrogen before harvesting.…”

“…Individual ERα/SRC-3 complexes were analyzed by the flow-proteometry system (Chou et al, 2014; Chou et al, 2016). In brief, ER (0.8 μg) plasmid DNA were transfected into HeLa cells (1×10 5 ) with or without CARM1 plasmid DNA (1.6 μg) using Lipofectamin 3000 (Life Technologies).…”

“…The lysates were then centrifuged 1 min at 16,000 × g and the supernatants were collected and further diluted in detection buffer (20 mM HEPES-KOH, pH 7.9, 0.1 mM KCl, 2 mM MgCl2, 15 mM NaCl, 0.2 mM EDTA, 10% (v/v) glycerol) for flow-proteometric analysis. The sample lysates were flowed in a microchannel device (Chou et al, 2014) and each ER/SRC-3 complex (SRC-3 labeled with Alexa 647 and ER labeled with Atto488) was detected by the flow-proteometric device (Chou et al, 2016). The fluorescence intensities of SRC-3 and ER in individual ER/SRC-3 complex were analyzed.…”