Résumé
Background
Neurofilament‐light chain (Nf‐L) is an early marker monitoring axonal damage which occur in many forms of dementia. During neurodegeneration, Nf‐L is released into biological fluid and is quantifiable in cerebrospinal fluid (CSF) and blood. Currently, its quantification is performed by immunoassays. While those ones are sensitive and precise, the development of a reference measurement procedure (RMP) using mass spectrometry (MS) approaches traceable to the International System of Units (SI) is essential to achieve standardisation.
Following the characterisation of three commercially available proteins as potential primary calibrator, one has been chosen and used for the method development. Here, we present the purity assessment and quantification traceable to the SI of the primary calibrator and preliminary data on the development of Nf‐L quantification in CSF by LC‐MS.
Method
A recombinant Nf‐L protein from Promise was characterized, SI traceably quantified via amino acid analysis and used as primary calibrator for the development of a LC‐MS method for Nf‐L. A triple quadrupole (QQQ) mass spectrometer coupled to a capillary liquid chromatography (LC) was used to set up a multiple reaction monitoring (MRM) method to monitor Nf‐L tryptic peptides and assess the potential of developing a RMP for Nf‐L.
Result
The homogeneity of Nf‐L from Promise was assessed on 3 aliquots prepared at 1.7 nmol/g. The mass fraction content in each vial was estimated at 1nmol/g ± 0.02 nmol/g.
A LC‐MRM method was developed to monitor Nf‐L tryptic peptides and assess their suitability for the development of a RMP. Fifteen peptides were selected. The limit of detection in solution is 1 ng/g for 6 peptides. The sample clean‐up was developed using Nf‐L spiked in artificial and real CSF. Several solid‐phase extraction cartridges were assessed in term of recovery and volume load. The use of protein precipitation was also assessed to increase the sensitivity. Preliminary experiments show a sensitivity of 100ng/g. Immunocapture will be used to achieve the required sensitivity.
Conclusion
A LC‐MRM based method and a sample clean‐up procedure were developed for the quantification of Nf‐L in CSF. A first assessment of sensitivity was carried out and the utilisation of immunocapture is on‐going to reach the required sensitivity.