Analysis of cerebrospinal fluid proteins by electrophoresis

The cerebrospinal fluid (CSF) is a specific ultrafiltrate of plasma, which surrounds the brain and spinal cord. The study of its proteins and their alteration may yield useful information on several neurological diseases. By using various electrophoretic separation techniques, several CSF proteins have been identified derived from plasma or from brain. Different one-dimensional methods, such as agarose gel electrophoresis and isoelectric focusing, are of similar value in identifying the non-specific oligoclonal bands, which are mainly helpful in the diagnosis of multiple sclerosis and other inflammatory diseases. Isoelectric focusing has a greater resolution than other one-dimensional methods, and it yields additional data about disease-associated proteins occurring in Alzheimer's disease, Huntington's chorea and amyotrophic lateral sclerosis. Silver-stained two-dimensional gels provide more information about the complex protein composition of CSF, particularly about proteins produced in the brain, such as apolipoprotein E and neuron-specific enolase. For the detection of oligoclonal antibodies, the investigation of protein changes revealed by Parkinson's disease, schizophrenia and Creutzfeldt-Jakob disease, and the analysis of CSF immune complexes, two-dimensional electrophoresis has a greater sensitivity.     

Identification of proteins from human cerebrospinal fluid, separated by two-dimensional polyacrylamide gel electrophoresis

The aim of this work is to display the protein composition of the cerebrospinal fluid by two-dimensional (2-D) gel electrophoresis and identify it using different mass spectrometric techniques. This will enable us to present an overview of the proteins in human cerebrospinal fluid. The comparison of 2-D gels will help us to analyze the normal protein variability in healthy persons and specific protein variations in patients with different neurological diseases (e.g., morbus Alzheimer, chorea Huntington). However, it is not possible to carry out 2-D gel electrophoresis directly with human cerebrospinal fluid due to the high amount of salts, sugars and lipids present. In addition, the total amount of protein is only as high as 0.3-0.7 microg/microL. Therefore, concentration and desalting steps using precipitation and ultrafiltration are necessary. To date we have been able to identify more than 65 spots from 2-D gels using matrix assisted laser desorption/ionization-mass spectrometry and electrospray ionization-mass spectrometry.     

Treponemal antigens detected by immunoblotting with serum and CSF antibodies of neurosyphilitic patients

Treponemal-antigen-eliciting antibodies in neurosyphilis were investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis immunoblotting with 17 sera and cerebrospinal fluid (CSF) (16 pairs) from 10 neurosyphilitic patients. Sera and CSF detected identical proteins. IgG antibodies in sera and CSF mainly revealed Treponema pallidum proteins of MW 48, 45, 38 and 37 Kd, and T. phagedenis proteins of MW 59, 54, 41, 40, 35 and 33 Kd. Few proteins were detected by IgM antibodies. Although no particularprotein elicited antibodies specific for neurosyphilis, the immunoblot detection of antibodies to T. pallidum or T. phagedenis antigens in CSF or sera should be useful as a diagnostic tool for neurosyphilis.     

Analysis of intact proteins from cerebrospinal fluid by matrix-assisted laser desorption/ionization mass spectrometry after two-dimensional liquid-phase electrophoresis

A novel combination of methods, two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), have been used for the analysis of intact brain-specific proteins in cerebrospinal fluid (CSF). 2D-LPE is especially useful for isolating proteins present in low concentrations in complex biological samples. The proteins are separated in the first dimension by liquid-phase isoelectric focusing (IEF) in the Rotofor cell and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the Preparative cell. The removal of SDS by chloroform/methanol/water, followed by sample preparation with the addition of n-octylglucoside, easily interfaced 2D-LPE with MALDI-TOFMS for analysis of intact proteins. Further characterization by proteolytic digestion is also demonstrated. The knowledge of both the molecular weights of the protein and of the proteolytic fragments obtained by peptide mapping increases specificity for protein identification by searching in protein sequence databases. Two brain-specific proteins in human CSF, cystatin C and transthyretin, were isolated in sufficient quantity for determination of the mass of the whole proteins and their tryptic digest by MALDI-TOFMS. This approach simplified the interface between electrophoresis and MALDI-TOFMS.     

Analysis of the human lumbar cerebrospinal fluid proteome

Idiopathic low back pain has no known cause, and the molecular basis is unknown. Neuropeptidergic systems have been previously studied, and proteomics methods have been applied in this present study. Proteomics combines high-resolution two-dimensional (2-D) gel electrophoresis, high-sensitivity mass spectrometry, and continuously expanding protein databases. Proteomics offers a comprehensive, bird's-eye view to analyze, at a systems level, all of the proteins in cerebrospinal fluid (CSF) that might contribute to idiopathic low back pain. CSF contains a high salt concentration and low protein concentration. In order to obtain a high-quality 2-D pattern, several sample preparation methods were tested to remove salts - protein precipitation with either acetone or trichloroacetic acid/acetone, or sample treatment with a Bio-Spin column. More spots were visualized on the 2-D gel of human CSF, and a relatively high protein recovery was obtained when a Bio-Spin column was used to process a human CSF sample. Sixty-one protein spots, obtained from 2-D gels with a pH range of either 3-10 or 4-7, were identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and MALDI-post-source decay (PSD)-MS. These 61 protein spots represent 22 proteins; six of those proteins were not annotated in any previously published 2-D maps. Those six proteins are PRO2619, pigment epithelium-derived factor, albumin homolog, kallikrein-6 precursor, DJ717I23.1, and AMBP protein precursor. These protein-mapping data will contribute to the database that will be used in the future to compare the proteomes obtained from the CSF of controls and low back pain patients, to characterize differentially expressed proteins, and to elucidate the biological markers for idiopathic low back pain.     

Application of high performance liquid chromatography combined with diode-array detection for analysis of proteins and peptides in human cerebrospinal fluid

Different strategies for HPLC separation, including molecular sieving, ion-exchange, and hydrophobic interaction as well as reversed phase chromatography, were used to study molecular components in human cerebrospinal fluid (CSF). The separations were followed by photodiode-array UV detection, which is a recently developed technique allowing a direct and rapid discrimination between peptides and proteins differing in their content of aromatic amino acids. By the various HPLC techniques in conjunction with diode-array detection it was possible to identify and characterize several protein and peptide components present in CSF. The procedure also allowed quantitative analysis of CSF proteins using minute amounts of the fluid.     

The separation and detection of alkaline oligoclonal IgG bands in cerebrospinal fluid using immobilised pH gradients

A method is described for the separation and detection of highly alkaline IgG bands in unconcentrated cerebrospinal fluid (CSF). These bands are frequently found in the cerebrospinal fluid of patients with inflammatory diseases of the central nervous system, particularly in the case of multiple sclerosis, and their detection is an important aid in clinical diagnosis. An isoelectric focusing technique using an immobilised pH gradient in polyacrylamide gel has been developed over the pH range 7-10, producing a linear and stable pH gradient with excellent resolution. After electrofocusing, the protein patterns were blotted onto polyvinylidene difluoride membranes and visualised using anti-human IgG followed by an enzyme-labelled second antibody. Blotting could be carried out by capillary diffusion for up to 16 h duration without any loss in resolution. Using this method, highly alkaline intrathecal IgG bands were found in the cerebrospinal fluid of all of the 14 multiple sclerosis patients. There were also 2 patients with alkaline IgG bands in their cerebrospinal fluid who were not diagnosed as multiple sclerosis. By contrast, no alkaline IgG bands with an isoelectric point (pI) greater than 8.6 were found in any of the serum samples studied (n = 50) from patients with various neurological disorders including multiple sclerosis.     

Identification of the apolipoprotein E4 isoform in cerebrospinal fluid with preparative two-dimensional electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

Apolipoprotein E (apoE) was isolated from human cerebrospinal fluid (CSF) from control individuals and patients with Alzheimer's disease (AD). The purification was performed with preparative two-dimensional electrophoresis (2-DE), involving liquid-phase isoelectric focusing (IEF) in the Rotofor cell in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution in the Mini Whole Gel Eluter. ApoE was characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of tryptic digests. The known change of Cys to Arg in position 112 of the apoE4 isoform was identified. This was detected in CSF from AD patients, reflecting the increased frequency of the apoE4 allele in this population. This peptide was not detected in CSF samples from healty control individuals. The use of this rapid electrophoretic separation in proteomic studies of CSF proteins provides single proteins, such as apoE, of high purity in yields sufficient for characterization by MALDI-TOF-MS. Characterization of proteins and their modifications (amino acid substitutions, glycosylation or phosphorylation) in CSF will be a useful tool in the investigation of the pathophysiology of brain disorders such as AD.     

Review on metal speciation analysis in cerebrospinal fluid-current methods and results: a review

The large number of patients suffering from neurodegenerative diseases like Alzheimer's disease and Parkinson's disease motivates many research groups worldwide to investigate pathogenic factors and molecular mechanisms of these diseases. Recent studies and reviews indicate that metals are involved in these neurodegenerative processes in case their homeostasis in the brain is disturbed. Important is that the focus of these recent studies is on essential metals like Fe, Cu, Zn and Mn, but not on the well-known neurotoxic metals like Hg and Pb. Key issues for understanding metal induced neurotoxic effects are the transport processes across the neural barriers, the metal binding forms (species) and their interactions with neuronal structures. Total metal concentrations in cerebrospinal fluid were published in several studies for controls and patients, but the amount of reliable data sets is not yet sufficient for clear definition of normal and elevated levels. The need for more detailed information on metal species in CSF is highlighted in this review. However, studies on element speciation analysis, that means identification and quantification of the various binding forms of metals in cerebrospinal fluid, are rare. The major reasons therefore are difficulties in accessing cerebrospinal fluid samples, the non-covalent nature of many metal species of interest and their rather low concentrations. In spite of this, several applications demonstrate the potential of hyphenated techniques as additional diagnostic tools for cerebrospinal fluid analysis. This review shows the importance of trace element analysis and more specifically of element speciation in cerebrospinal fluid for an improved understanding of pathologic mechanisms promoting neuro-degeneration. Respective analytical techniques are also highlighted. Additionally, biochemical assays for selected high molecular mass metal species are summarized and critically discussed. Moreover additional potential techniques like direct non-invasive methods as well as mathematical modelling approaches are considered. Data on total concentrations of numerous elements in CSF as well as speciation information of elements such as Al, As, Ca, Cd, Cu, Fe, Mg, Mn, Hg, Pb, Se and Zn in CSF are summarized.     

The simplified technique of high resolution two-dimensional polyacrylamide gel electrophoresis: biomedical applications in health and disease

The application of our simplified technique of high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to human body fluids is reviewed. Serum/plasma protein changes associated with alcohol abuse, familial dyslipoproteinemia ("fish-eye" disease), and myocardial infarction are demonstrated. High resolution 2-D PAGE of amniotic fluid, cerebrospinal fluid, urine, and saliva is shown with reference to the work of others, and the detection of pink-violet staining "lumicarmines" in sweat and tear fluid is reported for the first time. General aspects relating to the methodology are discussed. These include sample preparation, the choice of electrophoresis conditions (denaturing or nondenaturing) and detection method (Coomassie Brilliant Blue or silver), and the effects of native protein pretreatment with sodium dodecyl sulfate prior to silver staining or isoelectric focusing gel shrinkage in glycerol prior to second-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Recent Electrokinetic and Microfluidic Strategies for Detection of Amyloid Beta Peptide Biomarkers: Towards Molecular Diagnosis of Alzheimer's Disease

Among all neurodegenerative diseases, Alzheimer's Disease (AD) is the most prevalent worldwide, with a huge burden to the society and no efficient AD treatment so far. Continued efforts have been being made towards early and powerful diagnosis of AD, in the hope for a successful set of clinical trials and subsequently AD curative treatment. Towards this aim, detection and quantification of amyloid beta (Aβ) peptides in cerebrospinal fluid (CSF) and other biofluids, which are established and validated biomarkers for AD, have drawn attention of the scientific community and industry over almost two decades. In this work, an overview on our major contributions over 15 years to develop different electrokinetic and microfluidic strategies for Aβ peptides detection and quantification is reported. Accordingly, discussions and viewpoints on instrumental and methodological developments for microscale electrophoresis, microfluidic designs and immuno‐enrichment / assays on magnetic beads in microchannels for tracing Aβ peptides in CSF are given in this review.     

Comparison of oligoclonal immunoglobulin G bands in multiple sclerosis cerebrospinal fluid by immunoblotting and immunofixation

We previously analyzed unconcentrated cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and other neurologic diseases by isoelectric focusing in agarose gel. We have now developed an immunoblot method for detection of oligoclonal IgG bands in unconcentrated MS CSF. The oligoclonal IgG band patterns seen after immunoblotting were compared with those of conventional immunofixation. Although immunoblotting was found to be rapid the resolution and intensity of oligoclonal IgG bands were somewhat better after immunofixation. Since immunofixation is simpler than immunoblotting, we recommend that clinical laboratories use immunofixation after isoelectric focusing to detect oligoclonal IgG bands in CSF.     

Rapid determination of acyclovir in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection and its clinical application

A simple MEKC with UV detection at 254 nm for analysis of acyclovir in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of acyclovir from biological matrix was performed at 25 degrees C using a BGE consisting of Tris buffer with SDS as the electrolyte solution. Several parameters affecting the separation of the drug from biological matrix were studied, including the pH and concentrations of the Tris buffer and SDS. Using dyphylline as an internal standard, the linear ranges of the method for the determination of acyclovir in plasma and in CSF all exceeded the range of 2-50 microg/mL; the detection limit of the drug in plasma and in CSF (S/N = 3; injection 3.45 kPa, 5 s) was 1.0 microg/mL. The applicability of the proposed method for determination of acyclovir in plasma and CSF collected at 8 h after intravenous administration of 500 mg acyclovir (Zovirax) in two patients with herpes simplex encephalitis was demonstrated.     

Measurement of carnosine, homocarnosine and anserine by FASI capillary electrophoresis UV detection: applications on biological samples

A field amplified sample injection (FASI) capillary electrophoresis method with UV detection was developed for the separation and detection of carnosine-related peptides carnosine (Car), anserine (Ans) and homocarnosine (Hcar). The imidazole dipeptides were baseline-separated within 10 min by using 50 mmol/L Tris phosphate pH 2.2 as running buffer. The samples were diluted in water and directly injected on the capillary without complex cleanup and/or sample derivatization procedures. Using the electrokinetic injection, a sensitivity improvement of about 500-fold was achieved without any loss of separation efficiency if compared to the conventional sample injection. The detection limits for carnosine, anserine, and homocarnosine were between 0.4 and 0.5 nmol/L, thus improving of 10-100-fold the LOD of previous described methods based on laser induced fluorescence or chemiluminescence detection. This method has been applied to the analysis of homogenized rat tissue (heart, muscle and brain) and human cerebrospinal fluid (CSF).     

Determination of ceftazidime in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection

A simple micellar electrokinetic chromatography (MEKC) with UV detection at 254 nm for analysis of ceftazidime in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of ceftazidime from biological matrix was performed at 25 degrees C using a background electrolyte consisting of Tris buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Several parameters affecting the separation of the drug from biological matrix were studied, including pH and concentration of the Tris buffer and SDS. Using cefazolin as an internal standard (IS), the linear ranges of the method for the determination of ceftazidime in plasma and in CSF were all over the range of 3-90 microg/mL; the detection limit of the drug in plasma and in CSF (signal-to-noise ratio = 3; injection 0.5 psi, 5 s) was 2.0 microg/mL. The applicability of the proposed method for determination of ceftazidime in plasma and CSF collected after intravenous administration of 2 g ceftazidime in patients with meningitis was demonstrated.     

Sodium dodecylsulfate capillary gel electrophoretic measurement of the concentration ratios of albumin and alpha2-macroglobulin in cerebrospinal fluid and serum of patients with neurological disorders

Sodium dodecylsulfate capillary gel electrophoresis (SDS-CGE) was applied to measure the concentration ratios of albumin (Alb) and alpha2-macroglobulin (alphaMG) in the cerebrospinal fluid (CSF) and concurrent serum samples from patients with various neurological disorders. The values of the alphaMG index in individual patients were calculated on the basis of the peak area ratios of Alb and an alphaMG subunit on the CSF and serum electropherograms. The alphaMG index value thus obtained was most prominently raised in patients with inflammatory diseases of the brain and/or meninges, suggesting that the function of the blood-brain barrier (BBB) was disturbed under the pathological conditions in the central nervous system. The measurement of the concentration ratios of Alb and alphaMG in CSF and the concurrent serum samples by the present SDS-CGE system seems to be useful as an aid in the biochemical examination of the BBB function in patients with neurological disorders.     

Enzymic determination of cerebrospinal fluid lipid fractions

Colorimetric peroxidase-coupled procedures for the determination of several cerebrospinal fluid (CSF) lipid classes are described. These methods were modified to increase the effectiveness of each cerebrospinal fluid lipid assay by using the sample as the primary diluent for a highly concentrated reagent in an inverse concentration technique. Direct enzymic assays for the determination of CSF cholesterol (free and total), choline phospholipids, and triglycerides were adapted from existing assays to require less than 0.5 ml of sample per assay. This made determinations of the several lipid analytes possible even when samples were from pediatric specimens. In a study model, 51 pediatric CSF samples were analyzed for these lipid constituents. Mean values and standard deviations were determined. Within and between-run studies were performed by sampling from a pool of cerebrospinal fluid specimens. Within-run coefficients of variation for the several proposed procedures were less than 3% while the between-run findings for all of the procedures were less than 5%.     

Analysis of circulating immune complexes isolated from plasma, cerebrospinal fluid and urine.

The protein nature of soluble immune complexes (IC) from fresh plasma, cerebrospinal fluid (CSF), and urine was studied by combining several analytical and biochemical techniques. In plasma and CSF, free immunoglobulins G were separated from larger IC by gel filtration with a fast protein liquid chromatographic system. In urine, IC were separated by precipitation with polyethylene glycol. IC were further purified by protein-A and protein-G affinity chromatography and analyzed by two-dimensional gel electrophoresis. Apart from plasma samples from healthy donors, IC from cases with macrocreatine kinase type 1 and multiple sclerosis were analyzed. For CSF two cases of multiple sclerosis and for urine one case with urinary tract infection are shown. The method can be used for the examination of IC of unknown protein composition in body fluids.     

Horizontal two-dimensional electrophoresis of cerebrospinal fluid proteins with immobilized pH gradients in the first dimension.

A horizontal two-dimensional electrophoresis method with immobilized pH gradient isoelectric focusing supplemented with carrier ampholytes in the first dimension was applied to cerebrospinal fluid (CSF) proteins. About 300 protein spots could be detected on the silver-stained two-dimensional maps of CSF samples. This high-resolution method is a tool worthy of consideration for the research of CSF proteins and disease-specific changes in different neurological disorders.     

Rapid determination of piracetam in human plasma and cerebrospinal fluid by micellar electrokinetic chromatography with sample direct injection

A simple micellar electrokinetic chromatography (MEKC) method with UV detection at 200 nm for analysis of piracetam in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of piracetam from biological matrix was performed at 25 degrees C using a background electrolyte consisting of Tris buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Several parameters affecting the separation of the drug from biological matrix were studied, including the pH and concentrations of the Tris buffer and SDS. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Using imidazole as an internal standard (IS), the linear ranges of the method for the determination of piracetam in plasma and in CSF were all between 5 and 500 microg/mL; the detection limit of the drug in plasma and in CSF (signal-to-noise ratio=3; injection 0.5 psi, 5s) was 1.0 microg/mL. The applicability of the proposed method for determination of piracetam in plasma and CSF collected after intravenous administration of 3g piracetam every 6h and oral administration 1.2g every 6h in encephalopathy patients with aphasia was demonstrated.