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HPLC-MS/MS analysis of the S-monooxygenated S-Trp (O=S-Trp) bearing peptide in heme reconstituted EcKatG S-Trp105

ABSTRACT: A sulfur-containing analog of tryptophan, thiotryptophan (3-benzothienyl-L-alanine, S-Trp), was genetically incorporated into EcKatG to replace the MYW-forming tryptophan, W105. The heme free apo-protein was purified, after heme-reconstitution and peroxide treatment, the S-Trp-bearing peptide was isolated using trypsin digestion followed by HPLC separation. Tandem mass spectrometry (MS/MS) was then performed to verify the substitution. The final results demonstrated that S-Trp was successfully incorporated and subsequently underwent S-monooxygenation, yielding O=S-Trp.

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ORGANISM(S): Escherichia Coli

SUBMITTER: Ran Duan Duan

LAB HEAD: Aimin Liu

PROVIDER: PXD078069 | Pride | 2026-05-13

REPOSITORIES: Pride

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Project description:Scope: As a result of population ageing, the number of Alzheimer’s disease (AD) patients has rapidly increased. There are many hypothesises on the pathogenesis of AD, but its detailed molecular mechanism is still unknown, and so no effective preventive or therapeutic measures have been established. Some reports showed a decrease in levels of norepinephrine (NE) has been suspected to be involved in the decline of cognitive function in AD patients and NE concentrations were decreased in postmortem AD patient brains. Tyr-Trp was identified as being the most effective dipeptide in enhancing norepinephrine (NE) synthesis and metabolism. And Tyr-Trp treatment ameliorated the short-term memory dysfunction in AD model mice caused by amyloid beta (Aβ) 25-35. So, the purpose of this study was to investigate the preventive or/and protective effects of Tyr-Trp administration in AD model mice. Methods and results: ddY mice were fed normal diet. After 7 days of feeding, mice were divided into 3 groups (n=10 per group) as follows: the sham group, which received saline administration and distilled water injection; the Aβ group, which received saline administration and Aβ peptides 25–35 injection; and the Aβ+YW group, which received Tyr-Trp administration and Aβ peptides 25–35 injection. Tyr-Trp was orally administered Tyr-Trp (100 mg kg-1 day-1, twice a day), starting 7 days before Aβ peptides injection. The other groups received oral administration of saline (5 mL kg-1). After Aβ peptides injection or sham operation, each groups received every oral administration. DAN microarray showed that Tyrosine hydroxylase, dopa decarboxylase and dopamine receptor D2 mRNA expressions which were downregulated in Aβ group comparing with sham group were upregulated in Aβ+YW group comparing with Aβ group. In addition, quinoid dihydropteridine reductase mRNA expression which wasn’t changed in Aβ group comparing with sham group was upregulated in Aβ+YW group comparing with Aβ group. Conclusions: These results suggest that Tyr-Trp administration might ameliorate the short-term memory dysfunction in AD model mice because of enhancing norepinephrine (NE) synthesis and metabolism through metabolism of both Tyr and Trp.

Project description:Natural compounds that can stimulate salivary secretion are of interest in developing treatments for xerostomia, the perception of a dry mouth, that affects between 10 and 30% of the adult and elderly population. Chemesthetic transient receptor potential (TRP) channels are expressed in the surface of the oral mucosa. The TRPV1 agonists capsaicin and piperine have been shown to increase salivary flow when introduced into the oral cavity but the sialogogic properties of other TRP channel agonists have not been investigated. In this study we have determined the influence of different TRP channel agonists on the flow and protein composition of saliva. Mouth rinsing with the TRPV1 agonist nonivamide or menthol, a TRPM8 agonist, increased whole mouth saliva (WMS) flow and total protein secretion compared to unstimulated saliva, the vehicle control mouth rinse or cinnamaldehyde, a TRPA1 agonist. Nonivamide also increased the flow of labial minor gland saliva but parotid saliva flow rate was not increased. The influence of TRP channel agonists on the composition and function of the salivary proteome was investigated using a multi-batch quantitative mass spectrometry method novel to salivary proteomics. Inter-personal and inter-mouth rinse variation was observed in the secreted proteomes and, using a novel bioinformatics method, inter-day variation was identified with some of the mouth rinses. Significant changes in specific salivary proteins were identified after all mouth rinses. In the case of nonivamide, these changes were attributed to functional shifts in the WMS secreted, primarily the over representation of salivary and non-salivary cystatins which was confirmed by immunoassay. This study provides new evidence of the impact of TRP channel agonists on the salivary proteome and the stimulation of salivary secretion by a TRPM8 channel agonist, which suggests that TRP channel agonists are potential candidates for developing treatments for sufferers of xerostomia

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Dataset Information

HPLC-MS/MS analysis of the S-monooxygenated S-Trp (O=S-Trp) bearing peptide in heme reconstituted EcKatG S-Trp105

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