Feasible and reproducible synthesis of full-length Aβ peptides has been one of the major challenges in Alzheimer's disease research. By using dimethyl sulfoxide as an anti-aggregation solvent and as an agent to promote double-coupling of two phenylalanine that frequently experience residual deletion, we developed a reliable manual Fmoc solid phase peptide synthesis procedure to produce biologically active Aβ in large quantities at relatively high purity. The amyloidogenic activity of the synthesized Aβ was confirmed via thioflavin T assay, transmission electron microscopic analysis and electrophoresis.
The method was validated and applied to real samples in accordance with the external quality assessment scheme of WADA and to the previously reported samples that had provided positive test results. This novel method using hybrid sample preparation and LC/MS could be useful to screen multiple classes of the 264 targeted substances in anti-doping analysis.
Target analysis using liquid chromatography-tandem mass spectrometry is applied for rapidly detecting various prohibited doping substances. Frequent modification is required as additional substances are prohibited. We developed and validated a non-target screening method requiring no further modification because it analyzes the full spectrum of data in fixed m/z ranges. Urine samples were extracted using solid-phase extraction and analyzed by employing a method that combines full scan and variable data independent acquisition using high-resolution mass spectrometry; and all prohibited substances in the urine samples were successfully detected using our screening method. The method was validated in terms of specificity (no interferences), recoveries (29%-131%), matrix effects (35%-237%), limites of detection (0.0002-100 ng/mL), and intra- and inter-day precisions (coefficients of variation lower than 25%). The applicability of this method to doping tests was evaluated by analyzing 14 urine samples. As a result, the non-target screening method is efficient for conducting anti-doping tests because it can be applied without any further modification to prohibited drugs as well as to unknown targets that can be prohibited in the future.
One of the single nucleotide polymorphisms (SNPs) in human erythropoietin (hEPO), the c.577del variant, can produces 26 amino acids longer than the wild‐type hEPO, posing a risk of misinterpretation in routine doping analysis. To prevent this, the World Anti‐Doping Agency (WADA) included a procedure for reporting the sequencing results regarding the presence or absence of SNPs for suspected cases in the new version of the technical document for recombinant EPO in 2022. However, it is very expensive for anti‐doping laboratories to purchase a gene sequencing analyzer, which costs hundreds of thousands of dollars, and only a few companies provide specific gene sequencing services with accredited certification. Therefore, in this study, we developed a simple visualization method for the c.577del of the EPO variant at the gene level. The gene fragment of the EPO gene, including c.577del, was amplified using a fast polymerase chain reaction (PCR), and the PCR products were incubated with the clustered regularly interspaced short palindromic repeats (CRISPR)/deadCas9 system using variant‐specific single‐guide RNA (sgRNA). This ribonucleoprotein complex binds specifically to the EPO variant gene fragment, causing a band shift on native‐PAGE. We designed 4 sgRNAs that can bind only to the EPO variant or wild‐type gene. In addition, an electrophoretic mobility shift assay on a gel demonstrated that one of the sgRNAs had a high level of specificity. Consequently, the c.577del variant was selectively detected by visualizing the target fragment of the gene on the gel within 3 h using only 3 μl of the whole blood.
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