Quantitative analysis of amyloid-beta peptides in cerebrospinal fluid using immunoprecipitation and MALDI-Tof mass spectrometry.

Immunoprecipitation (IP) combined with matrix-assisted laser desorption ionization (MALDI) time of flight (Tof) mass spectrometry has been used to develop quantitative assays for amyloid-beta (Abeta) peptides in cerebrospinal fluid (CSF). Inclusion of (15)N labelled standard peptides allows for absolute quantification of multiple Abeta isoforms in individual samples. Characterization of variability associated with all steps of the assay indicated that the IP step is the single largest contributor to overall variability. Optimization of the assay resulted in overall coefficient of variation 相似文献   

Characterization and stability of transthyretin isoforms in cerebrospinal fluid examined by immunoprecipitation and high-resolution mass spectrometry of intact protein

Post-translational modifications (PTMs) contribute significantly to the complexity of proteins. PTMs may vary in certain patterns according to diseases and microenviroments making them potential markers for pathological processes. Human transthyretin (TTR) is a transporter of thyroxine and retinol in blood and cerebrospinal fluid (CSF). A single free cysteine thiol group in TTR possesses the ability to form mixed disulfides potentially related to diseases such as TTR amyloidosis and Alzheimer's disease (AD). Additionally, TTR-Cys10 S-thiolations might mirror the oxidative stress and redox balance of CSF. Here we describe a quick and gentle method for immunoprecipitating (IP) TTR from CSF with minimal introduction of sample-handling artifacts. A high-resolution mass spectrometer (LTQ-Orbitrap XL) was used in a simple setup with direct infusion that generates data suitable for confident assignment of TTR isoforms and validation of the protocol. Moreover, we demonstrate how simple storage of CSF at 4°C induces major oxidative modifications of TTR. Using the optimized method, we show data from a limited number of mild cognitive impairment (MCI) and AD patients. The protocol controls and minimizes the introduction of sample-handling artifacts during purification of TTR isoforms for high-resolution MS analysis.     

Identification of neuropeptide FF-related peptides in human cerebrospinal fluid by mass spectrometry

Several neuropeptide FF (NPFF)-related peptides, known as modulators of the opioid system, have been previously characterized in bovine and rodent brain. Reverse-phase high pressure liquid chromatography (HPLC) fractions of a human with normal pressure hydrocephalus cerebrospinal fluid (CSF), co-migrating with NPFF-related synthetic peptides, were characterized by capillary HPLC coupled on-line to nanospray ion trap tandem mass spectrometry. Two peptides present in the pro-NPFF(A) precursor, NPAF (AGEGLNSQFWSLAAPQRF-NH2) and NPSF (SLAAPQRF-NH2), were identified. The monitoring of NPFF-related peptides in human CSF can be helpful to understand their roles in pain sensitivity.     

Characterization of tau in cerebrospinal fluid using mass spectrometry

The neurodegenerative disorder Alzheimer's disease (AD) is the most common cause of dementia in the elderly. The presence of neurofibrillary tangles, consisting of hyperphosphorylated tau protein, is one of the major neuropathologic characteristics of the disease, making this protein an attractive biomarker for AD and a possible target for therapy. Here, we describe an optimized immunoprecipitation mass spectrometry method that enables, for the first time, detailed characterization of tau in human cerebrospinal fluid. The identities of putative tau fragments were confirmed using nanoflow liquid chromatography and tandem mass spectrometry. Nineteen tryptic fragments of tau were detected, of which 16 are found in all tau isoforms while 3 represented unique tau isoforms. These results pave the way for clinical CSF studies on the tauopathies.     

Molecular imaging of amyloid beta peptides in mouse brain sections using mass spectrometry

In this paper, a reverse-phase high-performance liquid chromatographic method using a chemically modified electrode coupled with microdialysis was developed to study the effect of electromagnetic impulse (EMI) on monoamine neurotransmitter metabolism in nerve cells. To detect the monoamines and their metabolites, a poly (para-aminobenzoic acid) (P-pABA)-modified electrode was prepared. The modified electrode exhibited efficiently electrocatalytic oxidation for monoamines and their metabolites with relatively high sensitivity, stability, and long life. Nerve cells were primarily cultured. EMI was radiated to three experimental model nerve cells: (i) on mature nerve cells, (ii) on the culture medium, and (iii) on juvenile nerve cells for various periods of time. Then the levels of monoamines in the culture medium were detected by high-performance liquid chromatography-electrochemical detection. The data indicated that electromagnetic fields could influence neurotransmitter metabolism by direct effect on nerve cells or effect on the nutrient medium and that the effect was not only relevant with the length of radiation time, but also with the growing state of the nerve cells.     

Peptide profiling of cerebrospinal fluid by mass spectrometry

The search for biomarkers is driven by the increasing clinical importance of early diagnosis. Reliable biomarkers can also assist in directing therapy, monitoring disease activity and the efficacy of treatment. In addition, the discovery of novel biomarkers might provide clues to the pathogenesis of a disease. The dynamic range of protein concentrations in body fluids exceeds 10 orders of magnitude. These huge differences in concentrations complicate the detection of proteins with low expression levels. Since all classical biomarkers have low expression levels (e.g., prostate-specific antigen: 2–4 µg/l; and CA125: 20–35 U/ml), new developments with respect to identification and validation techniques of the low-abundance proteins are required. This review will discuss the current status of profiling cerebrospinal fluid using mass spectrometry-based techniques, and new developments in this area.     

Peptide profiling of cerebrospinal fluid by mass spectrometry

The search for biomarkers is driven by the increasing clinical importance of early diagnosis. Reliable biomarkers can also assist in directing therapy, monitoring disease activity and the efficacy of treatment. In addition, the discovery of novel biomarkers might provide clues to the pathogenesis of a disease. The dynamic range of protein concentrations in body fluids exceeds 10 orders of magnitude. These huge differences in concentrations complicate the detection of proteins with low expression levels. Since all classical biomarkers have low expression levels (e.g., prostate-specific antigen: 2-4 microg/l; and CA125: 20-35 U/ml), new developments with respect to identification and validation techniques of the low-abundance proteins are required. This review will discuss the current status of profiling cerebrospinal fluid using mass spectrometry-based techniques, and new developments in this area.     

TOMM40 poly-T variants and cerebrospinal fluid amyloid beta levels in the elderly

A variable poly-T polymorphism in the TOMM40 gene, which is in linkage disequilibrium with APOE, was recently implicated with increased risk and earlier onset age for late-onset Alzheimer’s disease in APOE ε3 carriers. To elucidate potential neurobiological mechanisms underlying this association, we compared the effect of TOMM40 poly-T variants to the effect of APOE, an established LOAD-risk modulator, on cerebrospinal fluid (CSF) amyloid beta (Aβ) and tau levels, in cognitively intact elderly subjects. APOE ε4 carriers showed significant reductions in Aβ 1-42 levels compared to non-ε4 carriers, but no differences were detected across TOMM40 variants. Neither Aβ 1-40 nor tau levels were affected by APOE or TOMM40.     

Characterization of copper interactions with alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42

Cu and Zn have been shown to accumulate in the brains of Alzheimer's disease patients. We have previously reported that Cu(2+) and Zn(2+) bind amyloid beta (Abeta), explaining their enrichment in plaque pathology. Here we detail the stoichiometries and binding affinities of multiple cooperative Cu(2+)-binding sites on synthetic Abeta1-40 and Abeta1-42. We have developed a ligand displacement technique (competitive metal capture analysis) that uses metal-chelator complexes to evaluate metal ion binding to Abeta, a notoriously self-aggregating peptide. This analysis indicated that there is a very-high-affinity Cu(2+)-binding site on Abeta1-42 (log K(app) = 17.2) that mediates peptide precipitation and that the tendency of this peptide to self-aggregate in aqueous solutions is due to the presence of trace Cu(2+) contamination (customarily approximately 0.1 microM). In contrast, Abeta1-40 has much lower affinity for Cu(2+) at this site (estimated log K(app) = 10.3), explaining why this peptide is less self-aggregating. The greater Cu(2+)-binding affinity of Abeta1-42 compared with Abeta1-40 is associated with significantly diminished negative cooperativity. The role of trace metal contamination in inducing Abeta precipitation was confirmed by the demonstration that Abeta peptide (10 microM) remained soluble for 5 days only in the presence of high-affinity Cu(2+)-selective chelators.     

Detection of amyloid beta protein precursor immunoreactivity in normal and Alzheimer's disease cerebrospinal fluid

The amyloid A4 (or beta protein), a 4.2 kD polypeptide, is a major component of amyloid deposits in the brains of patients with Alzheimer's Disease (AD). The self-aggregating amyloid A4 protein of AD is encoded as part of three larger proteins by the amyloid A4 precursor gene. The corresponding proteins have 695, 751 and 770 amino acid residues. To investigate the utility of amyloid beta protein precursor (A beta PP) as a diagnostic marker for AD an antiserum against a synthetic peptide (175-186), predicted from cDNA sequence for A beta PP, was used. The immunoreactivity of A beta PP in normal and AD cerebrospinal fluid (CSF) was measured by Western blot and detected with radiolabeled protein A. A total of fifty-seven CSF samples (AD = 27 and normal = 30) were analyzed for A beta PP immunoreactivity. A polyclonal antibody detected two major protein bands with apparent molecular weights of 105kD and 90kD both in normal and AD CSF. The difference between normal and AD CSF was not significant. These results indicate that immunoreactivity of A beta PP is present both in normal and AD CSF, and that the difference is too small to be used as a diagnostic marker.     

Pro-opiocortin peptides in rat cerebrospinal fluid

Cerebrospinal fluid (CSF) taken from rats implanted with chronic cisternal cannulae was subjected to gel filtration chromatography on Sephadex G-50. Fractions were monitored using radioimmunoassays for N-terminal pro-opiocortin (N-POC), gamma 3-melanotropin (gamma 3-MSH), C-terminal adrenocorticotropin (C-ACTH), alpha-endorphin, beta-endorphin, gamma-lipotropin (gamma-LPH) and alpha-MSH. Two peaks which corresponded in elution position to rat N-POC (1-74) and gamma 3-MSH were detected. The major C-ACTH-immunoreactive (IR) peak was found to correspond to 14k ACTH. While no alpha-endorphin immunoreactivity was detected in rat CSF, three beta-endorphin-IR peaks were identified in positions expected for beta-LPH, beta-endorphin (1-31) and beta-endorphin (1-27), as well as a major peak of activity with the elution characteristics and cross-reactivity of rat gamma-LPH. HPLC of the alpha-MSH-IR material in rat CSF revealed the presence of a major peak of immunoreactivity whose retention time did not correspond to the known oxidised and reduced forms of alpha-MSH and its desacetylated and diacetylated derivatives. The identity of this peak is unknown.     

Structural analysis of 2D-gel-separated glycoproteins from human cerebrospinal fluid by tandem high-resolution mass spectrometry

The feasibility of global glycoprotein analysis by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry is demonstrated. Combined 2D gel glycoprotein separation and visualization, in-gel digestion, and accurate (<10 ppm) mass measurement allowed identification of human glycoproteins and revealed differences in glycosylation. IRMPD obviates the need for glycan release, which prevents sample dispersal, and allows the assignment of glycan structures to specific sites of N-glycosylation.     

Characterization of the oligomeric states of insulin in self-assembly and amyloid fibril formation by mass spectrometry

The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils.     

High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry

As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals.In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study.     

Xlink-identifier: an automated data analysis platform for confident identifications of chemically cross-linked peptides using tandem mass spectrometry

Chemical cross-linking combined with mass spectrometry provides a powerful method for identifying protein-protein interactions and probing the structure of protein complexes. A number of strategies have been reported that take advantage of the high sensitivity and high resolution of modern mass spectrometers. Approaches typically include synthesis of novel cross-linking compounds, and/or isotopic labeling of the cross-linking reagent and/or protein, and label-free methods. We report Xlink-Identifier, a comprehensive data analysis platform that has been developed to support label-free analyses. It can identify interpeptide, intrapeptide, and deadend cross-links as well as underivatized peptides. The software streamlines data preprocessing, peptide scoring, and visualization and provides an overall data analysis strategy for studying protein-protein interactions and protein structure using mass spectrometry. The software has been evaluated using a custom synthesized cross-linking reagent that features an enrichment tag. Xlink-Identifier offers the potential to perform large-scale identifications of protein-protein interactions using tandem mass spectrometry.     

Peptide mapping of proteins in cerebrospinal fluid utilizing a rapid preparative two-dimensional electrophoretic procedure and matrix-assisted laser desorption/ionization mass spectrometry

A quick two-step procedure involving liquid phase isoelectric focusing in the Rotofor cell in combination with electroelution in the Mini whole cell gel eluter has been used for purification of proteins from human cerebrospinal fluid (CSF). Fractions, each highly enriched in a single protein band and virtually free of other proteins, were selected for characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS). Six CSF proteins, transferrin, alpha1-acid-glycoprotein, Zn-alpha2-glycoprotein, apolipoprotein A1, apolipoprotein E and beta-trace were identified by MALDI-TOFMS analysis of the tryptic digests. These results demonstrate that the combination of liquid phase IEF and electroelution is a rapid preparative two-dimensional separation which can provide single proteins of high purity, in yields sufficient for characterization by MALDI-TOFMS. Characterization of such brain-specific proteins in CSF will be useful in the investigation of the pathophysiology of different brain disorders.