The signaling adaptor TRAF3 is a highly versatile regulator of both innate immunity and adaptive immunity, but how its phosphorylation is regulated is still unknown. Here we report that deficiency in or inhibition of the conserved serine-threonine kinase CK1ɛ suppressed the production of type I interferon in response to viral infection. CK1ɛ interacted with and phosphorylated TRAF3 at Ser349, which thereby promoted the Lys63 (K63)-linked ubiquitination of TRAF3 and subsequent recruitment of the kinase TBK1 to TRAF3. Consequently, CK1ɛ-deficient mice were more susceptible to viral infection. Our findings establish CK1ɛ as a regulator of antiviral innate immune responses and indicate a novel mechanism of immunoregulation that involves CK1ɛ-mediated phosphorylation of TRAF3.

To investigate the behavioral and biomolecular similarity between neuralgia and depression, a trigeminal neuralgia (TN) mouse model was established by constriction of the infraorbital nerve (CION) to mimic clinical trigeminal neuropathic pain. A mouse learned helplessness (LH) model was developed to investigate inescapable foot-shock-induced psychiatric disorders like depression in humans. Mass spectrometry was used to assess changes in the biomolecules and signaling pathways in the hippocampus from TN or LH mice. TN mice developed not only significant mechanical allodynia but also depressive-like behaviors (mainly behavioral despair) at 2 weeks after CION, similar to LH mice. MS analysis demonstrated common and distinctive protein changes in the hippocampus between groups. Many protein function families (such as cell-to-cell signaling and interaction, and cell assembly and organization,) and signaling pathways (e.g., the Huntington's disease pathway) were involved in chronic neuralgia and depression. Together, these results demonstrated that the LH and TN models both develop depressive-like behaviors, and revealed the involvement of many psychiatric disorder-related biomolecules/pathways in the pathogenesis of TN and LH.

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Due to the critical role glycation plays in many serious pathological conditions, such as diabetes, it is of great significance to discover protein glycation at an early stage for precaution and prediction of the disease. Here, a method of reductive amination combining dimethylation (RAD) was developed for the quantification of early-stage glycated proteins. The quantitative analysis was first carried out by reducing the samples using NaBHCN or NaBDCN, resulting in a 1 Da mass shift and the stabilization of early-stage protein glycation. The two samples were then digested and isotopically dimethylated to achieve the mass shift of 4 m + 3 n ( m represents the number of N-termini and Lys residues, and n represents the number of glycated sites) between light- and heavy-labeled glycated peptides for quantification. Consequently, the false positive result can be removed according to the different mass shifts of glycated peptides and non-glycated peptides. In quantification of glycated myoglobin, RAD showed good linearity ( R > 0.99) and reproducibility (CVs ≤ 1.6%) in 2 orders of magnitude (1:10-10:1). RAD was then applied to quantify the endogenous glycated proteins in the serum of diabetic patients, revealing significant differences in the glycation level between the patients with complicated retinal detachment and those without. In conclusion, RAD is an effective method for quantifying endogenous glycated proteins.

aims: In this work, we describe the development and interlaboratory co-validation of a middle-down UPLC-ToF MS MAM (multi-attribute method) analytical procedure for a fusion protein drug candidate CQAs control. background: Liquid chromatography mass spectrometry (LC-MS) is a good choice for the quality control of some biological product fragments which cannot be effectively controlled by traditional methods,such as CE-SDS, RP-HPLC. objective: To establish a new MS based MAM method for identity test and quantitatively monitor two critical quality attributes resulting from two truncations of that fusion protein. method: A subunit UPLC-ToF MS based MAM method was developed for identity test and quantitatively monitor two critical quality attributes resulting from two truncations of that fusion protein (fragment 1 and 2). Three independent laboratories are involved in the method validation according to ICH Q2(R1), ICH Q6B, FDA and NMPA guidance. result: Result shows that the developed method fully meet the pre-defined analytical target profile &(ATP&), including specificity, accuracy, precision, quantitation limit, linearity, range and robustness. conclusion: This is the first report describing the implementation of analytical method lifecycle management concept, and three independent labs co-validate a UPLC-ToF MS based MAM method for protein drug QC release and stability testing. The experimental design of method validation can be a reference for LC-HRMS based subunit MAM methods that have been being widely used in characterization of antibody, ADCs and other protein based biologics. This work paves the way for implementing MAM in QC with more targeted control of product quality. other: None