The effect of preanalytical factors on stability of the proteome and selected metabolites in cerebrospinal fluid (CSF).
To standardize the use of cerebrospinal fluid (CSF) for biomarker research, a set of stability studies have been performed on porcine samples to investigate the influence of common sample handling procedures on proteins, peptides, metabolites and free amino acids. This study focuses at the effect on proteins and peptides, analyzed by applying label-free quantitation using microfluidics nanoscale liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (chipLC-MS) as well as matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) and Orbitrap LC-MS/MS to trypsin-digested CSF samples. The factors assessed were a 30 or 120 min time delay at room temperature before storage at -80 degrees C after the collection of CSF in order to mimic potential delays in the clinic (delayed storage), storage at 4 degrees C after trypsin digestion to mimic the time that samples remain in the cooled autosampler of the analyzer, and repeated freeze-thaw cycles to mimic storage and handling procedures in the laboratory. The delayed storage factor was also analyzed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for changes of metabolites and free amino acids, respectively. Our results show that repeated freeze/thawing introduced changes in transthyretin peptide levels. The trypsin digested samples left at 4 degrees C in the autosampler showed a time-dependent decrease of peak areas for peptides from prostaglandin D-synthase and serotransferrin. Delayed storage of CSF led to changes in prostaglandin D-synthase derived peptides as well as to increased levels of certain amino acids and metabolites. The changes of metabolites, amino acids and proteins in the delayed storage study appear to be related to remaining white blood cells. Our recommendations are to centrifuge CSF samples immediately after collection to remove white blood cells, aliquot, and then snap-freeze the supernatant in liquid nitrogen for storage at -80 degrees C. Preferably samples should not be left in the autosampler for more than 24 h and freeze/thaw cycles should be avoided if at all possible.
Analytical Biochemistry, Department of Pharmacy, University of Groningen, Groningen, The Netherlands.
Therese
T
Rosenling
Christiaan L
CL
Slim
Christin
C
Christin
Leon
L
Coulier
Shanna
S
Shi
Marcel P
MP
Stoop
Jan
J
Bosman
Frank
F
Suits
Peter L
PL
Horvatovich
Norbert
N
Stockhofe-Zurwieden
Rob
R
Vreeken
Thomas
T
Hankemeier
Alain J
AJ
van Gool
Theo M
TM
Luider
Rainer
R
Bischoff
D000596:Amino Acids; D015415:Biomarkers / Q000134:cerebrospinal fluid; D002555:Cerebrospinal Fluid / Q000737:chemistry*; D015925:Cryopreservation; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000378:metabolism; D007962:Leukocytes / Q000737:chemistry / Q000378:metabolism; D054834:Lipocalins / Q000378:metabolism; D055432:Metabolomics; D010455:Peptides; D055550:Protein Stability; D011506:Proteins; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012015:Reference Standards; D013048:Specimen Handling / Q000379:methods* / Q000592:standards; D014021:Tissue Preservation / Q000379:methods* / Q000592:standards
D000596:Amino Acids; D015415:Biomarkers; D054834:Lipocalins; D010455:Peptides; D011506:Proteins; D020543:Proteome; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase