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µPhos: a scalable and sensitive platform for functional phosphoproteomics

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 in space and time is crucial for further illuminating functionality of the phosphoproteome. Here we describe µPhos (‘microPhos’), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture experiments in <8 hours total processing time. By minimizing transfer steps and reducing liquid volumes to <100 µL, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify >30,000 unique phosphopeptides in a human cancer cell line using 20 µg starting material, and confidently localize >10,000 phosphsites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable as we demonstrate by profiling the time-resolved response of leukemia cells to tyrosine kinase inhibitors.

INSTRUMENT(S): Bruker Daltonics timsTOF series

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Blood Cell

DISEASE(S): Chronic Myeloid Leukemia

SUBMITTER: Florian Meier

LAB HEAD: Jun-Prof. Florian Meier

PROVIDER: PXD043370 | Pride | 2024-05-27

REPOSITORIES: Pride

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			Action	DRS
	Denis_uPhos_mouse_brain_tissue.zip	Other
	Denis_uPhos_tissue.zip	Other
	ephos_volume_test.zip	Other
	ephos_volume_test_sne.zip	Other
	uPhos_CML_timeseries_ttest_tables.zip	Other

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Project description:Metformin, along with its biotransformation product guanylurea, are commonly observed in municipal wastewaters and subsequent surface waters. Previous studies in fish have identified metformin as a potential endocrine active compound but there are inconsistencies in the literature with regard to effects. To further investigate the potential reproductive toxicity of metformin and guanylurea to fish, a series of experiments were performed with reproductively mature fathead minnows (Pimephales promelas). First, explants of mature fathead minnow ovary tissue were exposed to 0.001-100 µM metformin or guanylurea to investigate whether they can directly perturb steroidogenesis. Second, spawning pairs of fathead minnows were exposed to metformin (0.41, 4.1, 41 µg/L) or guanylurea (1.0, 10, 100 µg/L) for 23 d to assess impacts on reproduction. Lastly, male fathead minnows were exposed to 41 µg/L metformin, 100 µg/L guanylurea, or a mixture of both compounds, with samples collected over a 96 h time course to investigate potential impacts to the hepatic transcriptome or metabolome. Neither metformin or guanylurea effected estradiol or testosterone by ovary tissue exposed in vitro. In the 23 d exposure, neither compound significantly impacted transcription of endocrine-related genes in male liver or gonad, circulating steroid concentrations in male or female fish, or fecundity of spawning pairs. In the 96 h time course, 100 µg guanylurea/L elicited more differential gene expression than 41 µg metformin/L , and showed the greatest impacts after 96 h. A number of DEGs up-regulated after 24 h were subsequently down-regulated after 96 h, demonstrating time-dependent impacts of guanylurea on the liver. Overall, metformin and guanylurea did not elicit effects consistent with reproductive toxicity in adult fathead minnows at environmentally relevant concentrations. Where effects were identified using ‘omics approaches, guanylurea induced greater impacts than metformin.

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µPhos: a scalable and sensitive platform for functional phosphoproteomics

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