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Updated: Jun 23, 2025
A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors
Published on: April 29, 2022
µPhos: a scalable and sensitive platform for high-dimensional phosphoproteomics
Denys Oliinyk1,2, Andreas Will1,2, Felix R Schneidmadel1,2
1Functional Proteomics, Jena University Hospital, 07747, Jena, Germany.
Abstract:
Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos ('microPhos'), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation.
Insights
Introducing µPhos (microPhos), a rapid phosphoproteomics platform enabling sensitive analysis of thousands of phosphorylation sites. This breakthrough facilitates detailed cellular response studies, even with limited samples.
Area of Science:
- Proteomics
- Cell Signaling
- Mass Spectrometry
Background:
- Phosphorylation analysis is key to understanding cell signaling.
- Current methods can be time-consuming and require large sample amounts.
- Defining cellular responses to perturbations requires sensitive phosphoproteomics.
Purpose of the Study:
- To develop an accessible and rapid phosphoproteomics platform (µPhos).
- To enhance sensitivity and quantitative reproducibility in phosphopeptide enrichment.
- To enable deep phosphoproteome analysis from limited sample quantities.
Main Methods:
- Developed µPhos (microPhos) for phosphopeptide enrichment from 96-well plates and small tissues.
- Minimized transfer steps and liquid volumes for increased efficiency.
- Utilized trapped ion mobility mass spectrometry for high-sensitivity quantification.
Main Results:
- µPhos achieves >90% selectivity and excellent quantitative reproducibility.
- Quantified ~17,000 Class I phosphosites from 20 µg and ~6200 from 1 µg of human cancer cell line.
- Revealed drug- and time-dependent response signatures in leukemia cells and spatial kinase activities in the mouse hippocampus.
Conclusions:
- µPhos significantly advances phosphoproteomics by enabling rapid, sensitive, and reproducible analysis.
- The platform supports sample-limited applications and large-scale perturbation studies.
- µPhos provides novel insights into cellular signaling and biological systems.
