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Edema factor (EF), a calmodulin-activated adenylyl cyclase, is a toxin which contributes to cutaneous and systemic anthrax. As a novel strategy to detect anthrax toxins in humans or animals infected by Bacillus anthracis, we have developed a sensitive enzymatic assay to be able to monitor functional EF in human and animal plasma. Samples containing EF are incubated in the presence of calmodulin and ATP, which is converted to cAMP. After oxidation and derivatization, cAMP is monitored by competitive enzyme immunoassay. Because of the high turnover of EF and the sensitivity of cAMP detection, EF can be detected at concentrations of 1 pg/ mL (10 fM) in 4 h in plasma from humans or at 10 pg/ mL in the plasma of various animal species using only a blood volume of 5 [micro]L. The assay has good reproducibility with intra- and interday coefficients of variation in the range of 20% and is not subject to significant interindividual matrix effects. In an experimental study performed in mice infected with the Berne strain, we were able to detect EF in serum and ear tissues. This simple and robust combination of enzymatic reaction and enzyme immunoassay for the diagnosis of anthrax toxemia could prove useful in biological threat detection as well in research and clinical practice.

In this study, we show that liquid chromatography coupled with tandem mass spectrometry provides a sensitive, specific, and accurate absolute quantification of Erbitux, a human:murine chimeric mAb used for the treatment of colorectal cancer. Micrometric magnetized beads, functionalized with soluble epidermal growth factor receptor (sEGFR), the pharmacological target of Erbitux, were used for specific immunocapture of Erbitux allowing assessment of the antibody's biological potency and sample purification. Following digestion with trypsin, specific peptides from light and heavy chains were monitored in the selected reaction monitoring (SRM) mode. Assay variability below 20% was provided through optimization of the digestion step and rigorous monitoring of the whole analytical process using an appropriate internal standard. The 20 ng/mL lower limit of quantification was similar to that of ELISA methods. These results show that this mass spectrometric approach is a potential alternative for pharmacokinetic evaluation of rnAbs during clinical development.

Direct LC/MS/MS methods have recently been developed for measuring triphosphate anabolites of several nucleosidic reverse transcriptase inhibitor (NRTI) in peripheral blood mononuclear cells (PBMCs) from HIV-positive patients. Whereas AZT is one of the most-used NRTIs, no such method has been developed for AZT-TP, its active anabolite, mainly because of the presence of endogenous nucleotides that interfere with such an assay. In this paper, we first describe the development of two enzyme immunoassays (EIA) of AZT-TP in PBMCs: one directly measuring AZT-TP content; the other, measuring the nucleoside AZT after selective extraction of AZT-TP and dephosphorylation. The precision of these two assays was too low to achieve precise determination of AZT-TP in PBMC samples. Direct LC/MS/MS is not specific enough for AZT-TP, since at least two interfering endogenous nucleotides (same m/z ratio and fragment as well as retention time close to that of AZT-TP) are found in the intracellular medium of PBMCs. The off-line combination of immunoaffinity extraction (IAE) and LC/MS/MS proved to be a successful strategy allowing without dephosphorylation appropriate specificity and sensitivity (limit of quantification established as 9.3 fmol/[10.sup.6] cells) to determine AZT-TP in PBMCs from 7 mL of blood of HIV-infected patients. Validation of this IAE-LC/MS/MS method demonstrated CV percent for repeatability and intermediate precision lower than 15%. More than 150 samples/ week can be analyzed by one analyst, making this method suitable for routine analysis during clinical studies.

Background: Neurodegenerative diseases diagnosis is still challenging, especially at the early stages. The diagnosis of Alzheimer's disease (AD) relies on the identification of tau proteoforms (i.e., phosphorylation) in the cerebrospinal fluid (CSF) and plasma. However, pathophysiological fluid biomarkers are still lacking for non‐AD tauopathies. Multiplex liquid chromatography (LC)‐mass spectrometry (MS/MS) offers the opportunity to quantify the vast diversity of tau proteoforms (3R and 4R isoforms, truncations, phosphorylations, acetylations). The current gold standard for tau extraction before LC‐MS/MS analysis of very low concentrations in biofluids relies on immunoprecipitation (IP) approaches. Nonetheless, IP may miss some proteoforms due to its high selectivity. Here, we tested anti‐body free and IP approaches and compared their ability to detect tau proteoforms with LC‐MS/MS. Method: We analyzed samples of artificial CSF (recombinant tau protein in diluted serum), and human CSF (from AD and non‐AD patients). Fully 15N‐labeled tau was added as an internal standard to the samples. Regarding IP, we first assessed a panel of commercial tau antibodies (in line with the literature) coated on magnetic beads. For the antibody‐free approach, we performed perchloric acid precipitation, followed by micro‐Solid Phase Extraction (Pc‐µSPE) using Oasis hydrophilic‐lipophilic balance sorbent. Purified proteins were digested into peptides using trypsin before multiplex quantitative analysis on reversed‐phase capillary LC, coupled with a qExactive high‐resolution mass spectrometer operated in the Parallel Reaction Monitoring (PRM) acquisition mode. Result: IP optimization led to a combination of antibodies targeting the central and N‐ter domains of tau. IP and Pc‐µSPE comparison in artificial CSF showed a higher sensitivity of IP: limits of detection decreased up to a factor 5. In the human CSF, Pc‐µSPE allowed for better detection of peptides from the N‐ter to the C‐ter than IP, and a higher signal for tau proteoforms (e.g., 3R and 4R isoforms and phosphorylated tau species). Conclusion: Despite better performances when dealing with canonical tau, IP was overcome by the antibody‐free sample preparation when identifying tau proteoforms with LC‐MS/MS in human CSF. The protocol is readily applicable to samples of clinical cohorts. This result opens the possibility of better detecting tau proteoforms and identifying pathophysiological biomarkers for non‐AD tauopathies. [ABSTRACT FROM AUTHOR]

Background: Tau protein aggregation into neurofibrillary tangles in the brain is a pathological hallmark of Alzheimer's disease (AD) and related tauopathies [1]. Though the mechanism of tau destabilization is not fully understood, tau protein exhibits several allele‐specific isoforms and post‐translational modifications (PTMs) specific to tau aggregates [2]. To explore in‐depth tau species in brain tissues of AD patients, we developed novel pipelines based on different tau fractions retrieved by buffer solubility, Filter Aided Sample Preparation (FASP) or immunoprecipitation, before high‐resolution mass spectrometry (HRMS) analysis. Method: As a proof of concept, we used frozen brain samples from two AD patients (0.6‐1.2 g per replicate, middle frontal gyrus). The samples were divided and homogenized, ultracentrifuged at 180.000xg for 30min and solubilized in different buffers successively (2 replicates): (1) Sark Method: low‐salt buffer, 10% Triton X‐100, sarkosyl buffer, and urea buffer; (2) RIPA Method: high‐salt buffer (twice), RIPA buffer and urea buffer; and, (3) GnH Method: guanidine hydrochloride and urea buffer. Then, we performed an antibody‐free approach by FASP or immunoprecipitation with a panel of commercial tau antibodies and analyzed on reversed‐phase capillary liquid chromatography, coupled with a qExactive HRMS. Result: HRMS demonstrated that the RIPA method performed better than the other methods for Tau analysis: coverage of 63% (full‐length, 2N4R) and 342 tau peptide‐spectrum matches (PSMs) (Sark: 49% and 183 PSMs; GnH: 41% and 169 PSMs). PTM‐modified tau species (phosphorylated, methylated, and acetylated) were primarily seen using the RIPA method (59 PSMs; Sark: 19 PSMs, GnH: 21 PSMs). Immunoprecipitation improved tau coverage and PTM detection compared to FASP. Conclusion: These findings highlight the best performance of the RIPA method followed by immunoprecipitation for the study of AD brain tau allele‐specific isoforms and PTMs using HRMS. Better characterization of Tau molecular profile in the brain of AD patients opens the gate for a better understanding of AD pathogenesis and represents an opportunity in the biomarker field. [1] Mroczko, B. et al. Int J Mol Sci 2019, 20, E4661, https://doi.org/10.3390/ijms20194661. [2] Boyarko, B.; Hook, V. Front. Neurosci. 2021, 15, 702788, https://doi.org/10.3389/fnins.2021.702788. [ABSTRACT FROM AUTHOR]