The Impact of Delayed Storage on the Measured Proteome and Metabolome of Human Cerebrospinal Fluid

Abstract

BACKGROUND Because cerebrospinal fluid (CSF) is in close contact with diseased areas in neurological disorders, it is an important source of material in the search for molecular biomarkers. However, sample handling for CSF collected from patients in a clinical setting might not always be adequate for use in proteomics and metabolomics studies. METHODS We left CSF for 0, 30, and 120 min at room temperature immediately after sample collection and centrifugation/removal of cells. At 2 laboratories CSF proteomes were subjected to tryptic digestion and analyzed by use of nano-liquid chromatography (LC) Orbitrap mass spectrometry (MS) and chipLC quadrupole TOF-MS. Metabolome analysis was performed at 3 laboratories by NMR, GC-MS, and LC-MS. Targeted analyses of cystatin C and albumin were performed by LC–tandem MS in the selected reaction monitoring mode. RESULTS We did not find significant changes in the measured proteome and metabolome of CSF stored at room temperature after centrifugation, except for 2 peptides and 1 metabolite, 2,3,4-trihydroxybutanoic (threonic) acid, of 5780 identified peptides and 93 identified metabolites. A sensitive protein stability marker, cystatin C, was not affected. CONCLUSIONS The measured proteome and metabolome of centrifuged human CSF is stable at room temperature for up to 2 hours. We cannot exclude, however, that changes undetectable with our current methodology, such as denaturation or proteolysis, might occur because of sample handling conditions. The stability we observed gives laboratory personnel at the collection site sufficient time to aliquot samples before freezing and storage at −80 °C.

GC/MS-based metabolomic analysis of cerebrospinal fluid (CSF) from glioma patients

“…Previous reports detected ~40–90 metabolites using GC/MS in normal human CSF [35–37], and the number of metabolites identified in this study was similar to that in the previous reports. The lactic acid level in the CSF was significantly elevated in the GBMs compared with the grades I–II gliomas.…”

Plasma and Cerebrospinal Proteomes From Children With Cerebral Malaria Differ From Those of Children With Other Encephalopathies

“…First, proteomic studies require careful sample collection and storage. Prolonged contact of CSF and plasma with cellular components has been shown to affect protein and peptide quality because of the presence of proteolytic enzymes in plasma [22, 23] and CSF [24, 25]. Samples analyzed in this study were centrifuged within an hour of collection to remove all cellular components, although protease inhibitors, which can minimize degradation, were not added.…”

Current trends and challenges in sample preparation for global metabolomics using liquid chromatography–mass spectrometry

“…For instance, residual enzymatic activity in refrigerated rat plasma resulted in increased levels of choline, glycerol, tyrosine, and phenylalanine [38]. In another study of the factors affecting the stability of metabolites in cerebrospinal fluid, the authors found that rapid centrifugation immediately (<5 min) after collection to remove white blood cells and snap-freezing (<2 h) were important to preserve accurate metabolite levels [39,40]. After delayed storage of 30 and 120 min, numerous metabolites (24 and 46 respectively, of a total 57 known metabolites detected) had significantly elevated levels, including glucose and ten amino acids (Thr, Glu, Gly, His, Ser, Ala, Phe, Tyr, Ile, and Asn), because of unquenched enzymatic activity, as shown in Fig.…”