Abstract
BackgroundVerification of cerebrospinal fluid (CSF) biomarkers for multiple sclerosis and other neurological diseases is a major challenge due to a large number of candidates, limited sample material availability, disease and biological heterogeneity, and the lack of standardized assays. Furthermore, verification studies are often based on a low number of proteins from a single discovery experiment in medium-sized cohorts, where antibodies and surrogate peptides may differ, thus only providing an indication of proteins affected by the disease and not revealing the bigger picture or concluding on the validity of the markers. We here present a standard approach for locating promising biomarker candidates based on existing knowledge, resulting in high-quality assays covering the main biological processes affected by multiple sclerosis for comparable measurements over time.MethodsBiomarker candidates were located in CSF-PR (proteomics.uib.no/csf-pr), and further filtered based on estimated concentration in CSF and biological function. Peptide surrogates for internal standards were selected according to relevant criteria, parallel reaction monitoring (PRM) assays created, and extensive assay quality testing performed, i.e. intra- and inter-day variation, trypsin digestion status over time, and whether the peptides were able to separate multiple sclerosis patients and controls.ResultsAssays were developed for 25 proteins, represented by 72 peptides selected according to relevant guidelines and available literature and tested for assay peptide suitability. Stability testing revealed 64 peptides with low intra- and inter-day variations, with 44 also being stably digested after 16 h of trypsin digestion, and 37 furthermore showing a significant difference between multiple sclerosis and controls, thereby confirming literature findings. Calibration curves and the linear area of measurement have, so far, been determined for 17 of these peptides.ConclusionsWe present 37 high-quality PRM assays across 21 CSF-proteins found to be affected by multiple sclerosis, along with a recommended workflow for future development of new assays. The assays can directly be used by others, thus enabling better comparison between studies. Finally, the assays can robustly and stably monitor biological processes in multiple sclerosis patients over time, thus potentially aiding in diagnosis and prognosis, and ultimately in treatment decisions.
Highlights
Verification of cerebrospinal fluid (CSF) biomarkers for multiple sclerosis and other neurological diseases is a major challenge due to a large number of candidates, limited sample material availability, disease and biological heterogeneity, and the lack of standardized assays
Full list of author information is available at the end of the article
There has been at least two publications going in the direction of generating high-quality parallel reaction monitoring (PRM) assays for CSF-proteins; one describing assays for 30 brain proteins [30], and another assays for monitoring a set of defined biological process [31]
Summary
Verification of cerebrospinal fluid (CSF) biomarkers for multiple sclerosis and other neurological diseases is a major challenge due to a large number of candidates, limited sample material availability, disease and biological heterogeneity, and the lack of standardized assays. JC viral antibody titers and neurofilament light, as summarized in a recent review [1] Both in-house discovery studies and available literature suggest numerous additional biomarkers representing several of the processes and pathways active in MS, such as inflammation and neurodegeneration [2,3,4,5]. The process of developing robust targeted assays in turn requires the consideration of a multitude of factors in order to ensure the quality and relevance of the assays, especially when the goal is to provide absolute protein measurements that would allow the consecutive analyses of proteins both across different labs and over time This will make it possible to monitor specific pathological processes occurring in individual MS patients and thereby gain a deeper insight into the processes active at the various stages of the disease, which in turn would be a valuable tool in diagnosis, prognosis and treatment decisions. It is crucial to create robust targeted assays for accurate measurements of biomarker candidates
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.