Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electophoresis approach prior to mass spectrometry

Davidsson, Pia; Paulson, Linda; Hesse, Camilla; Blennow, Kaj; Nilsson, Carol L.

doi:10.1002/1615-9861(200103)1:3<444::aid-prot444>3.0.co;2-q

Cited by 100 publications

(54 citation statements)

References 30 publications

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“…Typically, CSF samples showed a protein concentration ranging from 0.16 to 0.4 mg/mL. Aliquots containing 100 µg of total protein were taken from each sample and processed for 2DG analysis as follows 28,29 : samples were desalted against 10 mM Tris HCl pH 7 using p6 Bio-Spin columns (Bio-Rad). Each solution was then dried by centrifugation under vacuum.…”

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confidence: 99%

Sensitivity and specificity of decreased CSF asialotransferrin for eIF2B-related disorder

Vanderver¹,

Hathout²,

Maletkovic³

et al. 2008

Neurology

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Objective-This is a study estimating diagnostic accuracy of CSF asialotransferrin to transferrin ratio measurement in eIF2B related disorders by using clinical evaluation and EIF2B mutation analysis as the reference standard. eIF2B-related disorder is a relatively common leukodystrophy with broad phenotypic variation that is caused by mutations in any of the five EIF2B genes. There is a need for a simple and clinically valid screening tool for physicians evaluating patients with an unclassified leukodystrophy.Methods-CSF two-dimensional gel (2DG) electrophoresis analyses to measure asialotransferrin to transferrin ratios were performed in 60 subjects including 6 patients with documented EIF2B gene mutations, patients with other types of leukodystrophy, and patients with no leukodystrophy.Results-All six patients with mutation proven eIF2B-related disease showed low to nearly undetectable amounts of asialotransferrin in their CSF when compared to 54 unaffected controls by CSF 2DG analyses in this study. eIF2B-like patients, with clinically similar presentations but no mutations in EIF2B1-5, were distinguished from patients with mutations in EIF2B1-5 by this biomarker. Patients with mutations in EIF2B1-5 had asialotransferrin/transferrin ratio levels significantly different from the group as a whole (p < 0.001). Using 8% asialotransferrin/ transferrin ratio as a cutoff, this biomarker has a 100% sensitivity (95% CI = 52-100%) and 94% specificity (95% CI = 84-99%).Conclusion-Decreased asialotransferrin/transferrin ratio in the CSF of patients with eIF2B-related disorder is highly sensitive and specific. This rapid (<48 hours) and inexpensive diagnostic Copyright © 2008 eIF2B, composed of five subunits (eIF2Bα, eIF2Bβ, eIF2Bγ, eIF2Bδ, eIF2Bε), is encoded by five different genes (EIF2B1-5) with a total of 57 exons, making gene sequencing expensive and time consuming. Mutations have been found in all five genes and in multiple different exons. 6,25 The great number of individual mutations has confounded efforts at simplifying mutation analysis in suspected eIF2B-related cases, requiring in many cases the sequencing of all five genes to exclude a diagnosis.Efforts to establish a screening tool to streamline the diagnosis of eIF2B-related disorders have included CSF glycine levels, 26 measuring guanine nucleotide exchange factor activity of eIF2B in lymphoblasts (GEF activity), 27 and most recently, decreased CSF asialotransferrin to transferrin ratio. 28 Detection of CSF asialotransferrin to transferrin ratio by 2DG analysis has the advantage of being technologically simple, rapid (<48 hours), inexpensive, and highly sensitive. In this study, we focus on establishing the diagnostic accuracy of 2 DG analysis of CSF asialotransferrin to transferrin ratio for the diagnosis of eIF2B-related disorders. Current diagnosis is based on clinical evaluation followed by confirmation of eIF2B mutation, and this approach will be used as the reference standard. . CSF samples were studied in 60 subjects (figure 1): 6 patient...

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confidence: 99%

Sensitivity and specificity of decreased CSF asialotransferrin for eIF2B-related disorder

Vanderver¹,

Hathout²,

Maletkovic³

et al. 2008

Neurology

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“…Intact and biologically active nucleic acids and proteins have been obtained after post-mortem delays in excess of 72 h (Perrett et al 1988;Barton et al 1993;Leonard et al 1993;Cina 1994;Johnston et al 1997;Di Nunno et al 1998;Schramm et al 1999;Davidsson et al 2001;Schonberger et al 2001;Yasojima et al 2001;Bahn et al 2002;Johnson and Ferris 2002). Neither post-mortem interval (PMI) nor time in storage have been shown to significantly affect their integrity, although recoveries from human tissue can be highly variable (for a review see Barton et al 1993).…”

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confidence: 99%

“…Accurate and reproducible data can be obtained, provided a standardized system of brain collection, dissection, storage and case matching is employed in conjunction with the consideration of key ante-and post-mortem factors. Techniques in use in our and other laboratories range from quantitative competitive RT-PCR (Cummings et al 2001;Becker et al 2002;Guan et al 2002;Pandey et al 2002), western blotting (Lewohl et al 2001;Mori et al 2002), cDNA microarray (Whitney et al 1999;Lockhart and Barlow 2001;Goldstine et al 2002;Mayfield et al 2002;Tanwar et al 2002), and proteomic assays of unfixed tissue (Cheon et al 2001;Davidsson et al 2001;Husi and Grant 2001;Pasinetti 2001), through to RT-in situ hybridization histochemistry (Schindler et al 1995;Wakeman et al 1997;Hawkins and Dodd 2000;Goldstine et al 2002) and immunohistochemistry on fixed sections (Sparks et al 1998;Cerf and Raidoo 2000;Cervera et al 2002).…”

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confidence: 99%

Biochemical and molecular studies using human autopsy brain tissue

Hynd

Lewohl

Scott

et al. 2003

Journal of Neurochemistry

222

171

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The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable postmortem. However, their abundance and integrity can exhibit marked intra-and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante-and postmortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.

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“…As expected, many proteins were identified multiple times because of the great number of protein isoforms found in CSF. In another report, Davidsson et al used preparative liquid isoelectric focusing in combination with one-dimensional SDS gel electrophoresis for the identification of 32 CSF proteins, of which we identified 24 proteins in our analyses (14). This procedure was able to identify a number of low abundant proteins in undepleted CSF samples because of the concentration in a few fractions of the abundant HSA and Ig proteins during the preparative isoelectric focusing step.…”

Section: Human Csf Proteome _________________________________________mentioning

confidence: 73%

In-depth analysis of the human CSF proteome using protein prefractionation

et al. 2004

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The identification of disease markers in human body fluids requires an extensive and thorough analysis of its protein constituents. In the present study, we have extended our analysis of the human cerebrospinal fluid (CSF) proteome using protein prefractionation followed by shotgun mass spectrometry. After the removal of abundant protein components from the mixture with the help of immunodepletion affinity chromatography, we used either anion exchange chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to further subfractionate the proteins present in CSFs. Each protein subfraction was enzyme digested and analyzed by tandem mass spectrometry and the resulting data evaluated using the Spectrum Mill software. Different subfractionation methods resulted in the identification of a grant total of 259 proteins in CSF from a patient with normal pressure hydrocephalus. The greatest number of proteins, 240 in total, were identified after prefractionating the CSF proteins by immunodepletion and SDS-PAGE. Immuno-depletion combined with anion exchange fractionation resulted in 112 proteins and 74 proteins were found when only immunodepletion of the CSF sample was carried out. All methods used showed a significant increase in the number of identified proteins as compared with nondepleted and unfractionated CSF sample analysis, which yielded only 38 protein identifications. The present work establishes a platform for future studies aimed at a detailed comparative proteome analysis of CSFs from different groups of patients suffering from various psychiatric and neurological disorders.

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Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electophoresis approach prior to mass spectrometry

Cited by 100 publications

References 30 publications

Sensitivity and specificity of decreased CSF asialotransferrin for eIF2B-related disorder

Sensitivity and specificity of decreased CSF asialotransferrin for eIF2B-related disorder

Biochemical and molecular studies using human autopsy brain tissue

In-depth analysis of the human CSF proteome using protein prefractionation

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