µPhos: a scalable and sensitive platform for high-dimensional phosphoproteomics.
Denys OliinykAndreas WillFelix R SchneidmadelMaximilian BöhmeJenny RinkeAndreas HochhausThomas ErnstNina HahnChristian GeisMarkus LubeckOliver RaetherSean J HumphreyFlorian MeierPublished in: Molecular systems biology (2024)
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.
Keyphrases
- mass spectrometry
- endothelial cells
- single cell
- high resolution
- liquid chromatography
- induced pluripotent stem cells
- high throughput
- bone marrow
- protein kinase
- high performance liquid chromatography
- acute myeloid leukemia
- papillary thyroid
- capillary electrophoresis
- gas chromatography
- cell therapy
- gene expression
- ionic liquid
- squamous cell carcinoma
- pi k akt
- mesenchymal stem cells
- squamous cell
- blood brain barrier
- molecularly imprinted
- solid phase extraction
- drug induced
- childhood cancer
- simultaneous determination
- structural basis