Dataset Information

Effects of pro-opiomelanocortin neuropeptides on immunological pathways mediated by the melanocortin 3 receptor revealed by label-free quantitative thermal proteome profiling

ABSTRACT: In a thermal proteome profiling experiment we have identified 300 unique proteins that become thermally stabilized or destabilized downstream of signal activation of the melanocortin 3 receptor (MC3R) when expressed in HEK293 cells. Upon stimulation with either one of the endogenous MC3R-agonists ACTH, alpha-, or gamma-MSH, derived from the pro-opiomelanocortin (POMC) prohormone, we found that the majority of these thermally affected proteins is related to immunoregulatory processes including signaling cascades up- and downstream of the key transcription factors IRF, STAT and NFkappaB. Here, we have developed a computational pipeline that allows assessment of thermal melting curves from data obtained with label-free quantitation from ion-mobility enhanced LC-MS data. It enables differential expression analysis within the data obtained from a thermal proteome profiling experiment, since all relative abundance information is retained. We found with principal component analysis that the three POMC-peptides act in very distinguished ways on the expression pattern in the MC3R-expressing HEK293 cells. This is consistent with our findings from thermal proteome profiling analysis where we identified 142, 107 and 95 proteins to be thermally affected by ACTH, alpha- and gamma-MSH, respectively, with only 4 proteins identified across all three agonists, and an additional 36 proteins shared pairwise between the agonists. Interestingly, we found proteins involved in immune responses to be enriched according to gene set enrichment analysis with Reactome pathways. As a result of these findings, we inferred transcription factor activities of the involved thermally affected transcription factors using Bayesian analysis based on differential expression data. Our findings on transcription factor activity were validated with patterns of peptide phosphorylation abundance analysis. These combined efforts revealed that all three agonists act via the cAMP-PKA-CREB pathway but that there are differences in the further downstream affected pathways and the identities of the thermally affected pathways proteins in particular kinases.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Erik Jansson

LAB HEAD: Erik Jansson

PROVIDER: PXD039945 | Pride | 2023-10-24

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20210621_TPP_DMSO_37_001.mgf	Mgf
	20210621_TPP_DMSO_37_001.mzid.gz	Mzid
	20210621_TPP_DMSO_37_001.raw.zip	Raw
	20210621_TPP_DMSO_42_001.mgf	Mgf
	20210621_TPP_DMSO_42_001.mzid.gz	Mzid

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Publications

Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling.

Sandbaumhüter Friederike A FA Nezhyva Mariya M Andrén Per E PE Jansson Erik T ET

Analytical chemistry 20231007 41

Thermal proteome profiling with label-free quantitation using ion-mobility-enhanced LC-MS offers versatile data sets, providing information on protein differential expression, thermal stability, and the activities of transcription factors. We developed a multidimensional data analysis workflow for label-free quantitative thermal proteome profiling (TPP) experiments that incorporates the aspects of gene set enrichment analysis, differential protein expression analysis, and inference of transcript ...[more]

PMID: 37804223

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