Phospho-seq: Integrated, multi-modal profiling of intracellular protein dynamics in single cells

Abstract

Cell signaling plays a critical role in regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing, scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq, an integrated approach that aims to quantify phosphorylated intracellular and intranuclear proteins, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom antibody panels for simultaneous protein and scATAC-seq profiling on whole cells, and integrate this information with scRNA-seq datasets via bridge integration. We apply our workflow to cell lines, induced pluripotent stem cells, and 3-month-old brain organoids to demonstrate its broad applicability. We demonstrate that Phospho-seq can define cellular states and trajectories, reconstruct gene regulatory relationships, and characterize the causes and consequences of heterogeneous cell signaling in neurodevelopment.

Single-cell genotype-phenotype mapping identifies therapeutic vulnerabilities in VEXAS syndrome

“…Next, we sought to additionally measure single-cell intracellular protein levels together with genotype to directly validate PERK signaling in mutant cells. To this end, we integrated intracellular protein capture 26 into our GoT-ChA framework ( Fig. 5a ).…”

“…Here, to address outstanding mechanistic questions relating to VEXAS pathology and identify factors that could potentially be targeted for eradication of the disease-initiating HSCs, we apply multi-omic single-cell techniques, including Genotyping of Transcriptome (GoT) plus cellular indexing of transcriptomes and epitopes (CITE-seq) 25 and Genotyping of Targeted Loci with Chromatin Accessibility (GoT-ChA) 23 plus cell surface or intracellular (Phospho-Seq) 26 protein profiling by sequencing directly to VEXAS patient samples. High-resolution mapping of phenotypes across transcriptome, cell surface protein profiling, intracellular protein profiling and chromatin accessibility of UBA1 M41V/T vs. UBA1 wild-type cells shows mutation enrichment in myeloid cells, as well as in natural killer (NK) cells, which could be functionally impacted by the UBA1 M41V/T mutation.…”

CRISPR screening in hematology research: from bulk to single-cell level

“…These technologies remain under rapid development, with new applications such as profiling of the nascent transcriptome via PerturbSci-Kinetics, elucidating RNA dynamics [ 140 ]. Additionally, Phospho-seq enables the combination of scATAC with intracellular and intranuclear protein detection, with the possibility to integrate scRNA-seq data, combining three modalities within a single cell [ 141 ]. New single-cell technologies are being developed that do not require cell lysis for transcriptome analysis, thus keeping the cells alive and allowing temporal profiling of the same cells and studying trajectories [ 142 ].…”

The rise of single‐cell transcriptomics in yeast

“…However, these technologies remain unexplored in yeast. CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing), InCITE-seq (Intranuclear CITE-seq) and Phospho-seq simultaneously quantify extracellular, intranuclear, or phosphorylated epitopes within a scRNA-seq readout (Blair et al, 2023;Chung et al, 2021;Stoeckius et al, 2017). They use unique oligo-tagged antibodies to identify surface proteins, using sequencing as a readout.…”