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 degrees 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 micromol/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.
Bruegel
Mathias
M
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, Leipzig, Germany. mathias.bruegel@medizin.uni-leipzig.de
Planert
Mathis
M
Baumann
Sven
S
Focke
Almut
A
Bergh
Florian Then
FT
Leichtle
Alexander
A
Ceglarek
Uta
U
Thiery
Joachim
J
Fiedler
Georg Martin
GM
D015415:Biomarkers / Q000134:cerebrospinal fluid; D006801:Humans; D008280:Magnetics / Q000379:methods; D010455:Peptides / Q000134:cerebrospinal fluid*; D020543:Proteome / Q000378:metabolism*; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D015415:Biomarkers; D010455:Peptides; D020543:Proteome