Abstract
Mass spectrometry-based phosphoproteomic workflows enable routine study of signalling dynamics in contexts where cells are abundant. However, the analysis of cell signalling governed by phosphorylation requires an additional enrichment step which poses a significant challenge when dealing with limited material. The development of sensitive techniques that allow phosphoproteomic analysis of a few hundred cells is increasingly enabling researchers to disambiguate the complexity of protein signalling networks within heterogeneous cell populations. The imminent task at hand is to apply these techniques in research contexts guided by the available biological material to address complex questions about cellular function. Examples range from characterising differential treatment responses in distinct cell populations to investigating rare cell types from primary patient material or in vivo models. To achieve this, adapted protocols need to consider appropriate isolation of specific cells, simplified sample processing to avoid losses, labelling and multiplexing, and optimised analytical methodologies. Here, we discuss these aspects of the workflow, highlighting how innovations from low-input and single-cell proteomics can be adapted to drive low-input phosphoproteomics forward.