A candidate reference measurement procedure for the quantification of α-synuclein in cerebrospinal fluid using an SI traceable primary calibrator and multiple reaction monitoring.

α-synuclein aggregation is an important hallmark of neurodegenerative diseases such as Parkinson's disease (PD) and Lewy body dementia. α-synuclein has been increasingly used as a diagnostic biomarker in PD and other synucleinopathies. Current clinical assays rely on antibody-based immunoassays to detect α-synuclein, which possess high sensitivity, afford high throughput and require small sample volumes. The utility of these assays, however, may be compounded by the specificity, selectivity and batch-to-batch heterogeneity of the antibody used, which can lead to deviations in the total amount of the protein measured when comparing results among different laboratories. Similarly, current mass spectrometry-based quantification methods for α-synuclein lack well-defined, value assigned calibrators to ensure comparability of measurements. Therefore, there is still an unmet need for the standardisation of clinical measurements for α-synuclein that can be achieved by the development of reference measurement procedures (RMPs) utilising calibrators traceable to the SI (International System of Units). Here, we report a candidate RMP for α-synuclein, using an SI traceable primary calibrator and an isotope dilution mass spectrometry (IDMS) approach to address this need. The gravimetrically prepared primary calibrator was traceably quantified utilising a combination of amino acid analysis (AAA) and quantitative nuclear magnetic resonance (qNMR) for value assignment. An optimised targeted sample clean-up procedure involving a non-denaturing Lys-C digestion and solid-phase extraction strategy was devised, followed by the development of a targeted multiple reaction monitoring (MRM) method for the quantification of α-synuclein in cerebrospinal fluid (CSF). This candidate RMP was then deployed for the sensitive detection and accurate quantification of multiple proteotypic α-synuclein peptides in patient derived CSF samples. The LC-MS based results were subsequently compared to immunoassay data to assess the overall performance of our approach. The development and adoption of this candidate RMP, along with the availability of the SI traceable primary calibrator will allow for reliable quantifications of α-synuclein in CSF by an LC-MS based assay. The RMP will potentially contribute towards the standardisation of this important biomarker and may lead to future interlaboratory comparisons.

Results of the first and second British Mass Spectrometry Society interlaboratory studies on ambient mass spectrometry.

RATIONALE: As the popularity of ambient ionisation grows, so too does the importance of understanding its capabilities and limitations. The British Mass Spectrometry Society Special Interest Group on Ambient Ionisation has carried out two studies into the use of ambient ionisation, the results of which are presented here. METHODS: The first study (study 1) examined the detection and quantitation capabilities of ambient ionisation while the second examined repeatability and robustness. For study 1 participants were sent a range of samples including two calibration sample sets and asked to analyse them. For study 2, two samples containing the same eight-component mixture were provided (one in solvent, one in matrix); participants were asked to analyse these samples multiple times, over multiple days to allow assessment of repeatability. RESULTS: Study 1 showed that small, polar compounds were well detected by the participants while lower polarity compounds were less well detected. For many samples the introduction method appeared to be a significant factor in the observed spectra. The quantitation study gave good results but revealed significant variability. For study 2 the mean repeatabilities were 65% in solvent and 88% in matrix. The inclusion of an internal standard was shown to greatly improve repeatability. CONCLUSIONS: Ambient ionisation is capable of ionising a wide range of compounds with good precision and excellent repeatability; however, in order to obtain such data care must be taken with the experimental design. The data can be significantly improved with a well-chosen internal standard.

Almond or Mahaleb? Orthogonal Allergen Analysis During a Live Incident Investigation by ELISA, Molecular Biology, and Protein Mass Spectrometry.

It is now well known that an incident investigated in the United Kingdom in 2015 of cumin alleged to be contaminated with almond, a risk for people with almond allergy, was caused by the Prunus species, Prunus mahaleb. In the United Kingdom, the Government Chemist offers a route of technical appeal from official findings in the food control system. Findings of almond in two official samples, cumin and paprika, which had prompted action to exclude the consignments from the food chain, were so referred. Herein are described the approaches deployed to resolve the analytical issues during the investigation of the incidents. The cross-reactivity of ELISA to Prunus species was confirmed, and although this is useful in screening for the genus, orthogonal techniques are required to identify the species and confirm its presence. Two novel PCR assays were developed: one specific for P. mahaleb and the other a screening method capable of identifying common Prunus DNA. Peptides unique to almond and mahaleb were identified, permitting LC-tandem MS and criteria were developed for peptide identification to forensic standards. This work enables a staged approach to be taken to any future incident thought to involve Prunus species and provides a template for the investigation of similar incidents.