Abstract 1409 Using next-generation LC-MS to disentangle complex cell hierarchies

Single-cell proteomics by Mass Spectrometry (scp-MS) can provide valuable insights into distinct cell-states and signalling patterns present in a cell population. However, carrying out proteomics profiling from the limited amount of material encapsulated in an individual cell presents significant challenges. Tremendous efforts have been made to optimize all aspects of scp-MS, with the aim of minimizing losses during sample preparation and maximizing sensitivity of data acquisition. Here, we will present recent scp-MS approaches developed in the Cell Diversity Lab, ranging from TMTPro multiplexed assays to Data Independent Acquisition-based LFQ analysis of individual cells. We will cover key aspects of the various workflows, including cell sorting, sample preparation and chromatographic separation of extremely limited input material. Finally, we will showcase the application of our methods to biological questions spanning across stem cell differentiation and assessing the level of cell heterogeneity therein. With a particular focus on the healthy and malignant human blood system, we aim to convey possible biomedical implications of scp-MS, and the joint assessment of transcriptomes and proteomes at the single-cell level. Through these examples, we provide an overview of the current technological state of the field, and highlight key challenges that remain to be solved. This work was supported by generous funding from the Novo Nordisk Foundation, the LeoFoundation, the Danish Cancer Society, the Lundbeck Foundation, the Independent Research Fund Denmark, the Svend AndersenFoundation, and the Candys foundation.