The Effect of Preanalytical Factors on Stability of the Proteome and Selected Metabolites in Cerebrospinal Fluid (CSF)

Analytical Biochemistry, Department of Pharmacy, University of Groningen, Groningen, The Netherlands.
Journal of Proteome Research (Impact Factor: 4.25). 11/2009; 8(12):5511-22. DOI: 10.1021/pr9005876
Source: PubMed


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.

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    • "The LC–MS/MS method used for the measurement of plasma oxylipins and the LC–MS method used for the measurement of a broad range of plasma amino acids and derivatives were identical to the methods reported by, respectively, Balvers et al. (2012) and Rosenling et al. (2009). The samples were analyzed in randomized order. "
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    • "Our study is the first of its kind to test the stability of VEGF in aqueous humor samples and provides evidence of variation in VEGF measurement after 3 weeks of storage. This is consistent with results reported by Rosenling et al19 who observed a significant change in proteome-derived peptide profile in cerebrospinal fluid after storage for a week or 1 month, and with those of Aziz et al20 who reported a significant change in tumor necrosis factor alpha levels in plasma after storage, at 4°C or at −70°C for 20 days in comparison to samples stored at 24°C. This study’s results are also consistent with earlier study results on fresh versus frozen urine sample peptide concentration.21 "
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    • "Two studies performed in 2007 (Jimenez et al., 2007; Kaiser et al., 2007) analysed CSF with mass spectrometry and found no significant change in the CSF proteome when samples were kept at RT for 4–5 h. Rosenling et al. (2009) described a general change in the intensities of several peptides of the CSF proteome within the 2 h period before the storage of samples at −80 • C. The changes were pronounced 30 min after sampling. These results were more pronounced for samples that were analysed before centrifugation. "
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