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Photoaffinity for Puerarin and mouse brainstem

ABSTRACT: To elucidate the molecular mechanism mediating the inactivated effect of DMV neurons on fat absorption, we performed an activity-based protein profiling strategy, using Puerarin as a “bait”. The Puerarin-tag probe was synthesized with a photoreactive tag to enrich and visualize target proteins via a photoaffinity chemistry reaction. We verified that probe-tagged Puerarin retains the same effects of increasing fecal lipid excretion as non-tagged Puerarin. Probe-tagged Puerarin was added to the freshly isolated brainstem sample, and 10 doses of non-tagged Puerarin was used as a competitor of probe-tagged Puerarin. Following the photoaffinity reaction, targeted proteins were subsequently assessed by liquid chromatography tandem mass spectrometry (LC-MS).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Qianqian Lyu

LAB HEAD: Jiqiu Wang

PROVIDER: PXD052140 | Pride | 2024-09-06

REPOSITORIES: Pride

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Project description:Gametophytic self-incompatibility is the primary cause of low fruit set in almond. The mechanism of recognition that determines whether the gametophyte is successfully fertilized between pollen tube SCF (F-box-SSK1-Cul1-Rbx1) protein and pistil S-RNase protein during fertilization is unclear. In this study, the pistils of two almond cultivars 'Wanfeng' and 'Nonpareil' were used as the experimental materials after selfing- and nonselfing/cross-pollination, and pistils from the stamen-removed flowers were used as controls. We used fluorescence microscopy to observe the development of pollen tubes after pollination and 4-dimensional label-free quantitation (4D-LFQ) to detect the protein expression profiles of 'Wanfeng' and 'Nonpareil' pistils and in controls. The results showed that it took 24-36 h for the development of the pollen tube to 1/3 of the pistil, and a total of 7684 differentially accumulated proteins (DAPs) were identified in the pistil after pollinating 36 h, of which 7022 were quantifiable. The up- and down-regulation of the 12 differential protein expressions identified by parallel reaction monitoring (PRM) was the same as that identified by 4D-LFQ, with an average fold-change difference of 14.34%, a maximum of 31.37% and a minimum of 3.68%. Bioinformatics analysis based on the function of the differential proteins, including classification, enrichment, and clustering, identified RNA polymerases (4 DAPs), autophagy (3 DAPs), oxidative phosphorylation (3 DAPs), and homologous recombination (2 DAPs) pathways associated with the self-incompatibility process. The interaction between the serine/threonine kinase (MARK2) protein E3 ubiquitin ligase and the microtubule protein component microtubule-associated protein tau (MAPT/ACT) was found using the STRING database, which demonstrated the involvement of the MARK2 protein in the reaction of pollen tube recognition the nonself- and the self-S-RNase protein. It provides a new way to reveal the mechanism by which almond pollen tubes recognize the self and nonself S-RNase enzyme protein.

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Photoaffinity for Puerarin and mouse brainstem

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