Standardized peptidome profiling of human cerebrospinal fluid by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Peptidome profiling of human cerebrospinal fluid (CSF) is a promising tool to identify novel disease-associated biomarkers. Our aim was to develop a standardized protocol for reproducible peptidome profiling of CSF using magnetic bead (MB) separation followed by MALDI-TOF MS.Peptidome fractionation and profiling of CSF were performed using MBs with different surface functionalities. We investigated exogenous variables (storage conditions, freeze-thaw-cycles) and endogenous interferences (albumin, immunoglobulin, blood, leukocytes) in pooled CSF samples.We detected approximately 500 signals with an S/N ratio > 10 and an overlap frequency of about 40% in non-pathological CSF. Within- and between-day imprecisions in relative signal intensities ranged from 3 to 28% and 7 to 47%, respectively. CSF storage at room temperature for up to 6 h and at 4 °C for up to 3 days did not significantly influence the mass spectra. Consecutive freeze-thaw-cycles significantly affected the mass spectra. High albumin and immunoglobulin content altered the CSF preparation using MB-HIC C8 beads. Blood contamination showed no effect on mass spectra up to a hemoglobin concentration of 0.075 µmol/L. The presence of leukocytes up to a cell number of 30 Mpt/L did not affect mass spectra.Our reliable pretreatment protocol allows standardization of preanalytical modalities and thereby enables reproducible peptidome profiling of human CSF using MB separation followed by MALDI-TOF MS.