Here we present immuno-detection by sequencing (ID-seq) as a streamlined universal technology for measuring large numbers of molecular phenotypes, for many samples in parallel. In addition, several other recently described antibody– DNA conjugate-based methods that use high-throughput sequencing as a read-out detect only a few extracellular epitopes or at low sample throughput 8, 9, 10, 11, 12, limiting their scope. This assay is generally performed on cell lysates and currently there are no assays for phospho-proteins available to study signalling activity. Furthermore, 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. Although powerful, this strategy is not well suited for high-throughput applications. used the Nanostring system to quantify 88 antibody–single-strand DNA (ssDNA) oligo conjugates in fine needle aspirates 6.

Several alternative approaches based on antibody–DNA conjugates have been developed in recent years 6, 7. However, this approach requires cell lysis and does currently not include phospho-specific signalling detection. One commercial solution, Luminex ®, has circumvented this limitation by using colour-barcoded antibody-loaded beads and allows multiplexing of some 50 proteins per sample 3, 4, 5. However, these methods are limited in the number of (phospho-)proteins that can be measured simultaneously in each sample due to spectral overlap of the fluorescent reporter dyes. For instance, immunohistochemistry (IHC) 1 and immunofluorescence (IF) 2 allow high-throughput protein measurements using fluorescently labelled antibodies. Currently, a major limitation is the trade-off between the number of samples and the number of (phospho-)proteins that can be measured in a single experiment. However, the complexity of biological and disease processes is not easily captured by changes in individual markers.

This is particularly important for cell-based phenotypic screens to discover novel drug leads in the pharmaceutical industry. Quantification of protein levels and phosphorylation events is central to investigating the cellular response to perturbations such as drug treatment or genetic defects. Taken together, our work establishes ID-seq as a flexible solution for large-scale high-dimensional phenotyping in fixed cell populations. The results show an association between decreased mTOR signalling and increased differentiation and uncover 13 kinases potentially regulating epidermal renewal through distinct mechanisms. 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. Here we present the immuno-detection by sequencing (ID-seq) technology that combines antibody-based protein detection and DNA-sequencing via DNA-tagged antibodies. However, an accessible and universal technology for measuring large numbers of molecular and cellular phenotypes in many samples in parallel is not available. Scientists in the pharmaceutical industry as well as in academia have made tremendous progress in developing both large-scale and smaller-scale screening assays.

Cell-based small molecule screening is an effective strategy leading to new medicines.

Prota lyn review