Validation of an LC-MS/MS Method Using Solid-Phase Extraction for the Quantification of 1-84 Parathyroid Hormone: Toward a Candidate Reference Measurement Procedure.

peer reviewed[en] BACKGROUND: Parathyroid hormone (PTH) measurement is important for patients with disorders of calcium metabolism, including those needing bone-turnover monitoring due to chronic kidney disease-mineral bone disorder. There are currently 2 generations of PTH immunoassays on the market, both having cross-reactivity issues and lacking standardization. Therefore, we developed an LC-MS/MS higher-order method for PTH analysis. METHODS: The method was calibrated against the international standard for 1-84 PTH (WHO 95/646). Antibody-free sample preparation with the addition of an isotope-labeled internal standard was performed by solid-phase extraction. Extracts were analyzed by LC-MS/MS. EDTA-K2 plasma was used throughout the development and validation. Bias and uncertainty sources were tested according to ISO 15193. Clinical Laboratory Standards Institute guidelines and reference measurement procedures were consulted for the design of the validation. Patient samples and external quality controls were compared between LC-MS/MS and 2 third-generation immunoassays. RESULTS: The method was validated for 1-84 PTH from 5.7 to 872.6 pg/mL. The interassay imprecision was between 1.2% and 3.9%, and the accuracy ranged from 96.2% to 103.2%. The measurement uncertainty was <5.6%. The comparison between LC-MS/MS and the immunoassays showed a proportional bias but moderate to substantial correlation between methods. CONCLUSIONS: This LC-MS/MS method, which is independent of antibodies, is suitable for a wide range of PTH concentrations. The obtained analytical performance specifications demonstrate that development of a reference measurement procedure will be possible once a higher order reference standard is available

Modulation of αVβ6 integrin in osteoarthritis-related synovitis and the interaction with VTN (381-397 a.a.) competing for TGF-β1 activation

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USP8 Down-Regulation Promotes Parkin-Independent Mitophagy in the Drosophila Brain and in Human Neurons.

peer reviewedStress-induced mitophagy, a tightly regulated process that targets dysfunctional mitochondria for autophagy-dependent degradation, mainly relies on two proteins, PINK1 and Parkin, which genes are mutated in some forms of familiar Parkinson's Disease (PD). Upon mitochondrial damage, the protein kinase PINK1 accumulates on the organelle surface where it controls the recruitment of the E3-ubiquitin ligase Parkin. On mitochondria, Parkin ubiquitinates a subset of mitochondrial-resident proteins located on the outer mitochondrial membrane, leading to the recruitment of downstream cytosolic autophagic adaptors and subsequent autophagosome formation. Importantly, PINK1/Parkin-independent mitophagy pathways also exist that can be counteracted by specific deubiquitinating enzymes (DUBs). Down-regulation of these specific DUBs can presumably enhance basal mitophagy and be beneficial in models in which the accumulation of defective mitochondria is implicated. Among these DUBs, USP8 is an interesting target because of its role in the endosomal pathway and autophagy and its beneficial effects, when inhibited, in models of neurodegeneration. Based on this, we evaluated autophagy and mitophagy levels when USP8 activity is altered. We used genetic approaches in D. melanogaster to measure autophagy and mitophagy in vivo and complementary in vitro approaches to investigate the molecular pathway that regulates mitophagy via USP8. We found an inverse correlation between basal mitophagy and USP8 levels, in that down-regulation of USP8 correlates with increased Parkin-independent mitophagy. These results suggest the existence of a yet uncharacterized mitophagic pathway that is inhibited by USP8

Multidimensional performance assessment of micro pillar array column chromatography combined to ion mobility-mass spectrometry for proteome research

peer reviewedMicro pillar arrays columns (mPAC) are recent nanoflow liquid chromatographic (LC) systems featuring highly ordered pillars containing an outer porous shell grafted with C18 groups. This format limits backpressure and allows the use of extremely long separation channel (up to 2 m). In this study, we evaluated the use of mPAC in combination with ion mobility mass spectrometry (IM-MS). In IM-MS, ions are separated in gas-phase based on their size and charge. mPAC was compared to two other nanoflow systems and a state-of-the-art ultra-high-pressure liquid chromatograph (UHPLC). Performances in the four dimensions of information (LC, IM, MS and intensity) were calculated to assess the multidimensional efficiency of each tested system. mPAC proved to be superior to other nanoflow systems by producing more efficient peaks regardless of the gradient time employed which resulted in higher peak capacities (386 after 240 min gradient). In combination with IM, 3 times more peaks could be separated without loss of analysis time. Although UHPLC-ESI was superior from a chromatographic point of view, its sensitivity was rather limited compared to nanoflow LCs. On average, peaks in mPAC were 45-times more intense. Finally, mPAC combined to IM prove to enhance the proteome coverage by identifying two times more peptides than nanoflow LCs and ten times more than UHPLC. As a conclusion, mPAC combined to IM seems to be a suitable platform for discovery proteomics due to its high separation capacities

Interest of CE-IM-MS as a complementary tool to chromatographic-based methods for cell surface antigen discovery: proof-of-concept using human myeloma LP-1 cell line.

peer reviewedDisease management of multiple myeloma is challenging owing to patients’ multiple relapses and resistance to standard treatments. Therefore, the introduction of innovative cell therapies such as chimeric antigen receptor T-cells (CAR-T cells) opened new horizons in the outcome of severe refractory patients affected by aggressive forms of the disease. However, one of the limitations of the expansion of those treatments is the lack of specific MM tumor-associated antigens that could be targeted by current immunotherapies. Indeed, effectiveness of current CAR-T cells treatments could be hampered due to possible antigen-evasion strategies. Therefore, the discovery of new cell surface antigens could be an interesting approach to avoid occurrence of resistance that could lead to treatment failures. For this purpose, the use of mass spectrometry (MS) proteomics-based methodologies was considered. Due to the high sample complexity, liquid chromatography (LC) is commonly used prior MS detection to maximize protein identifications. Similarly, capillary electrophoresis (CE) could be an alternative due to its ability to provide high efficiency and high throughput separation. However, CE-MS performance could be impaired by lower sensitivity due to poor design of CE-MS interface as well as low sample loading capacity. In this study, those issues were tackled by using an online preconcentration technique namely dynamic pH junction to enhance sensitivity as well as loading capacity. Indeed, different conditions were compared in order to increase loaded volume without compromising separation efficiency. Besides, neutral-coated capillary was used in order to increase peak capacity leading to a higher number of identified entities compared to uncoated capillaries. Since CE separation principle is orthogonal to the mechanism that drives separation in LC, the use of both techniques to analyze the same sample allowed the increase of overall information in terms of number of identified proteins. Indeed, the interest of combining electrophoretic to chromatographic approaches was confirmed for the identification of antigens at the surface of human myeloma LP-1 cell line. As a matter of fact, despite the high number of proteins identified using LC-MS, more than a half of the proteins identified in CE could not be detected by LC. The capability of ion-mobility (IM) was also exploited in this study. Indeed, the addition of IMS module between LC or CE and MS lead to better proteome coverage due to the additional dimension of separation. To the best of our knowledge, little attention has been paid to the potential orthogonality between CE and IMS in proteomic studies to date. In this study the combination of CE with IMS allowed the separation of isobaric and co-migrating peptides leading to the identification of a larger number of unique proteins, thus increasing the possibility of detecting new antigens. In conclusion, the proof of concept concerning the interest of CE-IM-MS for the discovery of cell surface antigens was achieved during this study