Parallel reaction monitoring assay development for multiple sclerosis biomarkers in cerebrospinal fluid.
- Organism: Homo sapiens, Rattus norvegicus
- Instrument: Q Exactive HF
parallel reaction monitoring, cerebrospinal fluid, multiple sclerosis, neurological diseases, proteomics, biomarker
- Lab head:
We have developed and tested parallel reaction monitoring (PRM) assays for 25 proteins highly relevant as cerebrospinal fluid (CSF) biomarkers for multiple sclerosis, representing various biological processes affected in the disease. The proteins were represented by 72 peptides selected according to relevant guidelines and available literature. Stability testing revealed 64 peptides with low intra- and inter-day variations, with 44 also being stably digested after 16 hours of trypsin digestion, and 37 furthermore showing a significant difference between multiple sclerosis and controls, thereby confirming literature findings. Calibration curves were developed using spike-in of synthetic light and heavy peptides in rat plasma.
Assay peptides were thoroughly tested by parallel reaction monitoring (PRM) using heavy labelled peptides as internal standard. Potential peptides were tested for intra- and interday stability, digestion status after 1, 5, 16, 24 and 30 hours of trypsin incubation, and finally in a PRM study comparing 3 pools of multiple sclerosis patients to 3 pools of control patients. The peptides that passed all these test, were deemed most suitable as proteins surrogates and for monitoring various relevant processes in multiple sclerosis patients. Peptide calibration curves were developed in rat plasma by spike-in of constant heavy peptide and varying synthetic light peptide around the estimated concentration in CSF.
Cerebrospinal fluid from various patients. Collected by lumbar puncture. Rat plasma used for calibration curves.
Created on 1/24/20, 10:49 AM