Pre-analytical influence on the low molecular weight cerebrospinal fluid proteome.

Author(s): Berven FS, Kroksveen AC, Berle M, Rajalahti T, Flikka K, Arneberg R, Myhr KM, Vedeler C, Kvalheim OM, Ulvik RJ

Publication: Proteomics Clin Appl, 2007, Vol. 1, Page 699-711

PubMed ID: 21136725 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of filtration, centrifugation, and blood contamination on MALDI-TOF MS/MS analysis of CSF. For all experiments, aliquots of pooled CSF were used. For the blood contamination experiments, fresh whole blood from one healthy donor was added to pooled CSF aliquots.

   Summary of Findings:

   Filtration of CSF with either the 30-kDa Hydrosart or the 20-kDa cellulose triacetate MWCO filters was superior to other filter types for gel analysis. The 30-kDa poly(ethersulphone) filters gave no visible bands on Nu-PA gels, while the 10-kDa Hydrosart and 30-kDa Microcon MWCO filters had reproducibility issues. Slower centrifugation did not improve the reproducibility of the 10-kDa Hydrosart filters. Similar MALDI-TOF spectra were obtained for all filter types except for the 30-kDa poly(ethersulphone) filters which did not show some of the major peaks seen with other filters. The authors state that the use of guanidinium hydrochloride to break protein protein interactions was more efficient than using urea or 10 or 20% ACN. 0.02%. The authors also report that 0.1% blood contamination of CSF resulted in notable differences in spectra compared to no blood contamination when CSF was frozen without centrifugation to remove red and white cells prior to fractionation. However, the authors state that no differences were observed between contaminated and uncontaminated specimens when fractionation was performed immediately, or when centrifugation was used to remove red and white cells prior to freezing.

   Biospecimens

   • Bodily Fluid - Cerebrospinal Fluid
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified
   • Normal

   Platform:

   Analyte	Technology Platform
   Peptide	MALDI-TOF MS
   Peptide	1D/2D gels

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Biospecimen components	0 uL whole blood per mL CSF 0.2 uL whole blood per mL CSF 1 uL whole blood per mL CSF
   Biospecimen Aliquots and Components	Filtration	10-kDa cellulose triacetate MWCO filters 20-kDa cellulose triacetate MWCO filters 10-kDa Hydrosart filters 30-kDa Hydrosart MWCO spin filters 30-kDa Microcon MWCO filters 30-kDa poly(ethersulphone) filters
   Analyte Extraction and Purification	Protein solubilization	6 M Guanidinium hydrochloride 6 M urea 10% ACN 20% ACN None
   Biospecimen Aliquots and Components	Centrifugation	Centrifuged Not centrifuged

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2. Study Purpose

   The purpose of this study was to determine the effects of several storage parameters and the addition of protease inhibitors on MALDI-TOF MS/MS analysis of CSF. Individual specimens, rather than pooled CSF, were used for this study.

   Summary of Findings:

   No differences in CSF polypeptides detected in this study were found between specimens with protease inhibitor cocktail added prior to storage, prior to fractionation, or not at all. Storage of non-guanidinium-treated specimen fractions for 1 or 5 hours, at room temperature or on ice, before freezing had no effects on the spectra obtained. Frozen storage at -20 or -80 degrees C for 3 months, also had no effects other than reduced quality in the 1-2.5-kDa area due to contaminants compared to specimens stored at either temperature for 2 days. Two different peaks separated the spectra for the guanidinium fraction specimens stored at -80 degrees C (increased intensity of one peak) and -20 degrees C (appearance of an additional peak). Further, the guanidinium fraction specimens showed decreases in 2 peaks after storage at room temperature for 1 or 5 h or on ice for 1 h prior to frozen storage.

   Biospecimens

   • Bodily Fluid - Cerebrospinal Fluid

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Peptide	MALDI-TOF MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Protein solubilization	6 M Guanidinium hydrochloride None
   Storage	Storage temperature	Room temperature On ice -20 degrees C -80 degrees C
   Analyte Extraction and Purification	Protease inhibitor	Cocktail No protease inhibitor added Added before fractionation Added before storage
   Storage	Storage duration	0 h 1 h 5 h 2 d 3 months

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