Developing a Sensitive Platform to Measure 5-Methyltetrahydrofolate in Subjects with MTHFR and PON1 Gene Polymorphisms.

Inadequate levels of 5-methyltetrahydrofolate (5-MTHF) and the T variant of MTHFR C677T have been suggested to be associated with an increased risk of developing mental illness, whereas the PON1 SNP variant provides a protective role. However, reports validating the methodology for plasma 5-MTHF levels in schizophrenia patients are limited. A sensitive LC−MS/MS system using an amide column and calibration curve was determined by dialyzed human plasma, and applied to schizophrenia patients and healthy controls in Taiwan, and the differences between the subgroups were discussed. This analysis system meets regulation criteria, and the lower limit of quantification for 5-MTHF levels was 4 nM from 200 µL plasma, within 7 min. The mean plasma 5-MTHF levels in schizophrenia patients (n = 34; 11.70 ± 10.37 nM) were lower than those in the healthy controls (n = 42; 22.67 ± 11.12 nM) significantly (p < 0.01). 5-MTHF concentrations were significantly lower in male carriers than in female carriers (18.30 ± 10.37 nM vs. 24.83 ± 11.01 nM, p < 0.05), especially in subjects who were MTHFR CT/PON1 Q allele carriers. In conclusion, this quantitative system, which employed sensitive and simple processing methods, was successfully applied, and identified that schizophrenic patients had significantly lower levels of 5-MTHF. Lower plasma 5-MTHF concentrations were observed in male subjects.

Phosphoproteomics and Bioinformatics Analyses Reveal Key Roles of GSK-3 and AKAP4 in Mouse Sperm Capacitation.

Protein phosphorylation can induce signal transduction to change sperm motility patterns during sperm capacitation. However, changes in the phosphorylation of sperm proteins in mice are still incompletely understood. Here, capacitation-related phosphorylation in mouse sperms were firstly investigated by label-free quantitative (LFQ) phosphoproteomics coupled with bioinformatics analysis using ingenuity pathway analysis (IPA) methods such as canonical pathway, upstream regulator, and network analysis. Among 1632 phosphopeptides identified at serine, threonine, and tyrosine residues, 1050 novel phosphosites, corresponding to 402 proteins, were reported. Gene heatmaps for IPA canonical pathways showed a novel role for GSK-3 in GP6 signaling pathways associated with capacitation for 60 min. At the same time, the reduction of the abundant isoform-specific GSK-3α expression was shown by western blot (WB) while the LFQ pY of this isoform slightly decreased and then increased. The combined results from WB and LFQ methods explain the less inhibitory phosphorylation of GSK-3α during capacitation and also support the predicted increases in its activity. In addition, pAKAP4 increased at the Y156 site but decreased at the Y811 site in a capacitated state, even though IPA network analysis and WB analysis for overall pAKAP revealed upregulated trends. The potential roles of GSK-3 and AKAP4 in fertility are discussed.