Ikemoto, K. | Search bioRxiv

bioRxiv2025

Direct HPLC Method for Reduced Pyrroloquinoline Quinone in Functional and Biological Matrices

Pyrroloquinoline quinone (PQQ) is a redox-active compound with physiological functions, widely used in functional foods and supplements. However, quantifying the reduced form, PQQred, is difficult due to its low solubility and susceptibility to oxidation. This study presents a robust HPLC method for direct quantification of PQQred in complex matrices. By changing to a strongly acidic eluent, the oxidation of PQQred was suppress, and the optimized method successfully separated PQQred from its oxidized counterpart (PQQox)and matrix interferences, enabling accurate quantification. A pretreatment using ascorbic acid and {gamma}-CD effectively reduced and solubilized PQQred, and total PQQ can be analyzed as PQQred. The method achieved excellent linearity (R2 = 1), low detection limits (LOD: 0.20 mg/L, LOQ: 0.50 mg/L), and high precision (RSD < 3%). Application to commercial beverages showed consistent recovery (99-101%) with minimal interference. Moreover, the method suctandem mass spectrometry.cessfully detected biologically generated PQQred in yeast cultures, demonstrating its utility in physiological systems. This redox-specific and practical approach enables routine analysis and quality control of PQQ-enriched products, especially where accurate assessment of redox state is essential.

biochemistry↗

bioRxiv2022

Localized assembly for long reads enables genome-wide analysis of repetitive regions at single-base resolution in human genomes

BackgroundLong-read sequencing technologies have the potential to overcome the limitations of short reads and provide a comprehensive picture of the human genome. However, it remains hard to characterize repetitive sequences by reconstructing genomic structures at high resolution solely from long reads. Here, we developed a localized assembly method (LoMA) that constructs highly accurate consensus sequences (CSs) from long reads. MethodsWe first developed LoMA, by combining minimap2, MAFFT, and our algorithm, which classifies diploid haplotypes based on structural variants and constructs CSs. Using this tool, we analyzed two human samples (NA18943 and NA19240) sequenced with the Oxford Nanopore sequencer. We defined target regions in each genome based on mapping patterns and then constructed a high-quality catalog of the human insertion solely from the long-read data. ResultsThe assessment of LoMA showed high accuracy of CSs (error rate < 0.3%) compared with raw data (error rate > 8%) and superiority to the previous study. The genome-wide analysis of NA18943 and NA19240 identified 5,516 and 6,542 insertions ({zeta} 100 bp) respectively. Most insertions ([~]80%) were derived from the tandem repeat and transposable elements. We also detected processed pseudogenes, insertions in transposable elements, and long insertions (> 10 kbp). Further, our analysis suggested that short tandem duplications were association with gene expression and transposons. ConclusionsOur analysis showed that LoMA constructs high-quality sequences from long reads with substantial errors. This study revealed the true structures of insertions with high accuracy and inferred mechanisms for the insertions. Our approach contributes to the future human genome studies. LoMA is available at our GitHub page: https://github.com/kolikem/loma.

genomics↗