Chemical Methods for the Simultaneous Quantitation of Metabolites and Proteins from Single Cells

Abstract

We describe chemical approaches for integrated metabolic and proteomic assays from single cells. Quantitative assays for intracellular metabolites, including glucose uptake and 3 other species, are designed as surface-competitive binding assays with fluorescence readouts. This enables integration into a micro-array format with functional protein immunoassays, all of which are incorporated into the microchambers of a single-cell barcode chip (SCBC). Using the SCBC, we interrogate the response of human-derived glioblastoma cancer cells to EGFR inhibition. We report, for the first time, on both the intercellular metabolic heterogeneity as well as the baseline and drug-induced changes in the metabolite-phosphoprotein correlation network. Graphical AbstractThe emergence of powerful single-cell genomic, transcriptomic, and proteomic tools over the past decade has yielded exciting approaches towards resolving the heterogeneity of complex biological systems. [1][2][3] To date, most single cell tools have focused on transcriptome or proteome analysis, or on the sequencing of specific sets of genes. Quantitative single cell metabolic assays have proven more challenging, although there mass spectrometric methods are promising. [4][5][6] No reports on the integration of metabolite assays with other classes of Corresponding Author. ; Email: heath@caltech.edu (J.R.H.) # These authors contributed equally. ASSOCIATED CONTENT Supporting InformationSynthetic schemes, experimental details and statistical methods. This material is available free of charge via the Internet at http:// pubs.acs.org.The authors declare no competing financial interests. Quantitative measurements (generating copy numbers per cell) of intracellular proteins can be accomplished using calibrated, sandwich-type immunofluorescence assays. Such assays require a surface-bound capture antibody and a fluorophore-labeled detection antibody, and yield an optical readout that correlates with protein copy number. These assays can be miniaturized and multiplexed through spatial addressing using the single cell barcode chip (SCBC) format. Metabolites are small molecules, and so cannot be similarly detected by antibody pairs. We report on three types of spatially-addressable competition assays designed to measure the absolute or relative levels of 4 small molecule metabolites, in a manner that allows those assays to be integrated into SCBC (or other) proteomic assays. HHS Public AccessThe SCBC platform, the metabolite competition assays, and calibration and validation data are provided in Figure 1. The SCBC (Fig 1a) consists of 310 1.5 nanoliter microchambers into which cells are loaded, and each of which contains a full barcode array. Each microchamber has a companion lysis buffer reservoir separated by a programmable valve (Supporting Figure S1). 7,8 For protein assays, specific stripes in the barcode represent a spatial address upon which a sandwich immunofluorescence assay for a specific protein is executed. Each barcode stripe is initially patterned with a ...

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