Lydia Emilie Aakervik,a,c, Sofie Lysholm Lian,c,d, Bernd Thiede,e, Manuel Ramirez Garrastacho,e, Kaja
Nordengen,c and
Steven Ray Wilsona,b
a Section of Chemical Life Science, Department of Chemistry, University of Oslo, Norway
b Hybrid Technology Hub, Faculty of Medicine, University of Oslo, Norway
c Department of Neurology, Oslo University Hospital, Oslo, Norway
d Institute of Clinical medicine, Faculty of medicine, University of Oslo, Oslo, Norway
e Section of Biochemistry and Molecular Biology, Department of Biosciences, University of Oslo, Norway
Email: lydiaea@uio.no
Cerebrospinal fluid (CSF) is in direct contact with the central nervous system and can potentially reveal brain disorders, damages, and diseases (1). This study aims to develop a reliable and robust method for sample preparation of CSF for untargeted nano liquid chromatography-mass spectrometry (nanoLC-MS)-based proteomics.
The cohort for this study consists of 10 healthy controls and 21 Parkinson’s disease patients. CSF was prepared for nanoLC-MS analysis by protein precipitation, followed by trypsin digestion and desalting of resolubilized protein pellets. The sample preparation methods´ recovery and repeatability was critically evaluated by total protein/peptide content quantification and gel electrophoresis (SDS-PAGE).
Protein recovery after the precipitation procedure was measured to 80%. Based on visual inspection of the electrophoresis gel, trypsin digestion appeared successful, as most of the protein bands were no longer visible. Peptide recovery after desalting was heavily dependent on the applied SPE procedure, ranging from 20% to nearly 100%. However, we also found that available total peptide content measurements lack robustness, calling for improved recovery assays. As a final step in the method validation, a random CSF pool was prepared as described above and analyzed using nanoLC-MS. A total of 1463 proteins were identified, with a coefficient of variation of 6% across replicates (n=4), indicating repeatability. Among the 10 healthy controls, 1317 proteins were identified, with a coefficient of variation of 3% between the samples.
A method to evaluate the composition of proteins in CSF can provide insight in physiological and pathological states of the central nervous system (2), and we are currently applying the method to discover novel biomarkers.
References
1. Macron C, Lane L, Núñez Galindo A, Dayon L. Deep Dive on the Proteome of Human Cerebrospinal Fluid: A Valuable Data Resource for Biomarker Discovery and Missing Protein Identification. J Proteome Res. 2018;17(12):4113-26.
2. Begcevic I, Brinc D, Drabovich AP, Batruch I, Diamandis EP. Identification of brain-enriched proteins in the cerebrospinal fluid proteome by LC-MS/MS profiling and mining of the Human Protein Atlas. Clin Proteomics. 2016;13:11.