Abstract
Cell-based small molecule screening is an effective strategy leading to new medicines. Scientists in the pharmaceutical industry as well as in academia have made tremendous progress in developing both large-scale and smaller-scale screening assays. However, an accessible and universal technology for measuring large numbers of molecular and cellular phenotypes in many samples in parallel is not available. Here we present the immuno-detection by sequencing (ID-seq) technology that combines antibody-based protein detection and DNA-sequencing via DNA-tagged antibodies. We use ID-seq to simultaneously measure 70 (phospho-)proteins in primary human epidermal stem cells to screen the effects of ~300 kinase inhibitor probes to characterise the role of 225 kinases. The results show an association between decreased mTOR signalling and increased differentiation and uncover 13 kinases potentially regulating epidermal renewal through distinct mechanisms. Taken together, our work establishes ID-seq as a flexible solution for large-scale high-dimensional phenotyping in fixed cell populations.
Highlights
Cell-based small molecule screening is an effective strategy leading to new medicines
At the basis of immunodetection by sequencing (ID-seq) lie antibodies that are labelled with a double-stranded DNA tag[13] containing a 10-nucleotide antibody-dedicated barcode and a 15-nucleotide unique molecular identifier (UMI, Supplementary Fig. 1, Supplementary note 1)
DNA barcodes are released from the antibodies through reduction of a chemically cleavable linker[13] and a sample-specific barcode is added through PCR
Summary
Cell-based small molecule screening is an effective strategy leading to new medicines. Quantification of protein levels and phosphorylation events is central to investigating the cellular response to perturbations such as drug treatment or genetic defects This is important for cell-based phenotypic screens to discover novel drug leads in the pharmaceutical industry. Luminex®, has circumvented this limitation by using colour-barcoded antibody-loaded beads and allows multiplexing of some 50 proteins per sample[3,4,5] This approach requires cell lysis and does currently not include phospho-specific signalling detection. The commercial Proseek® strategy entails a proximity extension assay using pairs of ssDNA oligo coupled antibodies in combination with quantitative PCR as a read-out[7] This assay is generally performed on cell lysates and currently there are no assays for phospho-proteins available to study signalling activity. This demonstrates a downregulation of mammalian target-of-rapamycin (mTOR) signalling during differentiation and uncoveres 13 kinases potentially regulating epidermal renewal through distinct mechanisms
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