| 1. |
- Brinkmalm, Gunnar, et al.
(författare)
-
Soluble amyloid precursor protein α and β in CSF in Alzheimer's disease.
- 2013
-
Ingår i: Brain research. - : Elsevier BV. - 1872-6240 .- 0006-8993. ; 1513, s. 117-26
-
Tidskriftsartikel (refereegranskat)abstract
- Cerebral accumulation of amyloid β (Aβ) is a pathological hallmark of Alzheimer's disease (AD). Proteolytic processing of amyloid precursor protein (APP) by α- or β-secretase results in two soluble metabolites, sAPPα and sAPPβ, respectively. However, previous data have shown that both α- and β-secretase have multiple cleavage sites. The aim of this study was to characterize the C-termini of sAPPα and sAPPβ in cerebrospinal fluid (CSF) by mass spectrometry (MS) and to evaluate whether different combinations of these fragments better separate between AD patients and controls by comparing two different sAPP immunoassays. Methods: Using immunoprecipitation and high resolution MS, the APP species present in CSF were investigated. CSF levels of sAPPα and sAPPβ from patients with AD (n=43) and from non-demented controls (n=44) were measured using AlphaLISA and MSD immunoassays that employ different antibodies for C-terminal recognition of sAPPα. Results: Four different C-terminal forms of sAPP were identified, sAPPβ-M671, sAPPβ-Y681, sAPPα-Q686, and sAPPα-K687 (APP770 numbering). Neither immunoassay for the sAPP species could separate the two patient groups. The correlation (R(2)) between the two immunoassays was 0.41 for sAPPα and 0.45 for sAPPβ. Conclusion: Using high resolution MS, we show here for the first time that sAPPα in CSF ends at Q686 and K687. The findings also support the conclusion from several previous studies that sAPPα and sAPPβ levels are unaltered in AD.
|
|
| 2. |
- Augutis, Kristin, et al.
(författare)
-
Cerebrospinal fluid biomarkers of β-amyloid metabolism in multiple sclerosis.
- 2013
-
Ingår i: Multiple sclerosis (Houndmills, Basingstoke, England). - : SAGE Publications. - 1477-0970 .- 1352-4585. ; 19:5, s. 543-52
-
Tidskriftsartikel (refereegranskat)abstract
- Amyloid precursor protein (APP) and amyloid β (Aβ) peptides are intensely studied in neuroscience and their cerebrospinal fluid (CSF) measurements may be used to track the metabolic pathways of APP in vivo. Reduced CSF levels of Aβ and soluble APP (sAPP) fragments are reported in inflammatory diseases, including multiple sclerosis (MS); but in MS, the precise pathway of APP metabolism and whether it can be affected by disease-modifying treatments remains unclear.
|
|
| 3. |
- Haußmann, Ute, et al.
(författare)
-
Analysis of Amino-Terminal Variants of Amyloid-β Peptides by Capillary Isoelectric Focusing Immunoassay.
- 2013
-
Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 85:17, s. 8142-9
-
Tidskriftsartikel (refereegranskat)abstract
- Here we present a novel assay for the separation and detection of amino-terminal amyloid-β (Aβ) peptide variants by capillary isoelectric focusing (CIEF) immunoassay. Specific amino-terminally truncated Aβ peptides appear to be generated by β-secretase (BACE1)-independent mechanisms and have previously been observed in cerebrospinal fluid (CSF) after BACE1 inhibitor treatment in an animal model. CIEF immunoassay sensitivity is sufficient to detect total Aβ in CSF without preconcentration. To analyze low-abundance amino-terminally truncated Aβ peptides from cell culture supernatants, we developed a CIEF-compatible immunoprecipitation protocol, allowing for selective elution of Aβ peptides with very low background. CIEF immunoassay and immunoprecipitation mass spectrometry analysis identified peptides starting at residue Arg(5) as the main amino-terminal Aβ variants produced in the presence of tripartite BACE1 inhibitor in our cell culture model. The CIEF immunoassay allows for robust relative quantification of Aβ peptide patterns in biological samples. To assess the future possibility of absolute quantification, we have prepared the Aβ peptides Aβx-10, Aβx-16, and Aβ5-38(D23S) by using solid phase peptide synthesis as internal standards for the CIEF immunoassay.
|
|
| 4. |
- Krastins, B., et al.
(författare)
-
Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum
- 2013
-
Ingår i: Clinical Biochemistry. - : Elsevier BV. - 0009-9120. ; 46:6, s. 399-410
-
Tidskriftsartikel (refereegranskat)abstract
- Objectives The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. Design and methods The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. Results In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67–0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. Conclusions We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.
|
|
| 5. |
- Meredith, Jere E, et al.
(författare)
-
Characterization of novel CSF Tau and ptau biomarkers for Alzheimer's disease.
- 2013
-
Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 8:10
-
Tidskriftsartikel (refereegranskat)abstract
- Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer's disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.
|
|
| 6. |
- Nutu, Magdalena, 1967, et al.
(författare)
-
Aβ1-15/16 as a potential diagnostic marker in neurodegenerative diseases.
- 2013
-
Ingår i: Neuromolecular medicine. - : Springer Science and Business Media LLC. - 1559-1174 .- 1535-1084. ; 15:1, s. 169-79
-
Tidskriftsartikel (refereegranskat)abstract
- Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) reflect brain biochemistry. Using combined immunoprecipitation and mass spectrometry, we have shown that amyloid beta 1-15 (Aβ1-15) is produced by concerted β- and α-secretase cleavage of amyloid precursor protein (APP) and that the relative levels of Aβ1-16 in AD compared to controls are increased. Furthermore, drug-induced γ-secretase inhibition enhances the relative levels of Aβ1-15 and Aβ1-16. Here, we investigate a novel immunoassay for Aβ1-15/16 in a broad range of neurodegenerative conditions. The CSF level of Aβ1-15/16 was measured by the bead-based amplified luminescent proximity homogeneous assay (Alpha technology). Concentrations of Aβ1-15/16 were analyzed in subjects with Parkinson disease (PD; n = 90), PD with dementia (PDD) (n = 32), dementia with Lewy bodies (DLB) (n = 68), AD (n = 48), progressive supranuclear palsy (PSP) (n = 45), multiple system atrophy (MSA) (n = 46), and corticobasal degeneration (CBD) (n = 12). The detecting antibody is specific to the C-terminal epitope of Aβ15. We found that a carboxypeptidase (CPB) present in fetal bovine serum (FBS), a component of the buffers used, degrades Aβ1-16 to Aβ1-15, which is then detected by the Aβ1-15/16 assay. Significantly, lower levels of Aβ1-15/16 were detected in PD, PDD, PSP, and MSA compared to other neurodegenerative diseases and controls. Using the specific Aβ1-15/16 assay, a reliable quantification of Aβ1-15 or Aβ1-15/16 in CSF samples is obtained. We found reduced levels of Aβ1-15 in parkinsonian disease groups. The molecular mechanism behind this reduction is at present unknown.
|
|
| 7. |
- Pannee, Josef, 1979, et al.
(författare)
-
A selected reaction monitoring (SRM)-based method for absolute quantification of Aβ38, Aβ40, and Aβ42 in cerebrospinal fluid of Alzheimer's Disease patients and healthy controls.
- 2013
-
Ingår i: Journal of Alzheimer's Disease. - 1387-2877. ; 33:4, s. 1021-32
-
Tidskriftsartikel (refereegranskat)abstract
- Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in research centers, clinical trials, and clinical settings. However, their broad-scale use is hampered by lack of standardization across analytical platforms and by interference from binding of amyloid-β (Aβ) to matrix proteins as well as self-aggregation. Here, we report on a matrix effect-resistant method for the measurement of the AD-associated 42 amino acid species of Aβ (Aβ42), together with Aβ40 and Aβ38 in human CSF based on mass spectrometric quantification using selected reaction monitoring (SRM). Samples were prepared by solid-phase extraction and quantification was performed using stable-isotope labeled Aβ peptides as internal standards. The diagnostic performance of the method was evaluated on two independent clinical materials with research volunteers who were cognitively normal and AD patients with mild to moderate dementia. Analytical characteristics of the method include a lower limit of quantification of 62.5 pg/mL for Aβ42 and coefficients of variations below 10%. In a pilot study on AD patients and controls, we verified disease-association with decreased levels of Aβ42 similar to that obtained by ELISA and even better separation was obtained using the Aβ42/Aβ40 ratio. The developed assay is sensitive and is not influenced by matrix effects, enabling absolute quantification of Aβ42, Aβ40, and Aβ38 in CSF, while it retains the ability to distinguish AD patients from controls. We suggest this SRM-based method for Aβ peptide quantification in human CSF valuable for clinical research and trials.
|
|
| 8. |
- Portelius, Erik, 1977, et al.
(författare)
-
Mass spectrometric characterization of amyloid-β species in the 7PA2 cell model of Alzheimer's disease.
- 2013
-
Ingår i: Journal of Alzheimer's disease : JAD. - 1875-8908. ; 33:1, s. 85-93
-
Tidskriftsartikel (refereegranskat)abstract
- The Chinese hamster ovary cell line 7PA2, stably transfected with the 751 amino acid isoform of amyloid-β protein precursor (AβPP) containing the Val → Phe mutation at residue 717, is one of the most used models to study the biochemistry and toxicity of secreted amyloid-β (Aβ) peptides, particularly Aβ oligomers, which are considered to be of relevance to the pathogenesis of Alzheimer's disease. Here, we present a detailed immunochemical and mass spectrometric characterization of primary structures of Aβ peptides secreted by 7PA2 cells. Immunoprecipitation and western blot of 7PA2 cell culture media revealed abundant anti-Aβ immunoreactive bands in the molecular weight range of 4-20 kDa. Mass spectrometric analysis showed that these bands contain several AβPP/Aβ peptides, starting at the N-terminal of the Aβ sequence and extending across the BACE1 cleavage site. Treatment of cells with a BACE1 inhibitor decreased the abundance of the Aβ monomer band by western blot and resulted in lower levels of Aβ1-40, Aβ1-42, and sAβPPβ as measured by ELISA. However, western blot bands thought to represent oligomers of Aβ increased in response to BACE1 inhibition. This increase was paralleled by the emergence of N-terminally truncated Aβ species (Aβ5-40 in particular) and Aβ species that spanned the β-secretase site in AβPP according to mass spectrometric analyses. The formation of these AβPP/Aβ peptides may have implications for the use of the 7PA2 cell line as a model for Aβ pathology. The enzyme(s) responsible for this particular BACE1-independent AβPP-processing remains to be identified.
|
|
| 9. |
- Sitek, E. J., et al.
(författare)
-
A Patient with Posterior Cortical Atrophy Possesses a Novel Mutation in the Presenilin 1 Gene
- 2013
-
Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 8:4
-
Tidskriftsartikel (refereegranskat)abstract
- Posterior cortical atrophy is a dementia syndrome with symptoms of cortical visual dysfunction, associated with amyloid plaques and neurofibrillary tangles predominantly affecting visual association cortex. Most patients diagnosed with posterior cortical atrophy will finally develop a typical Alzheimer's disease. However, there are a variety of neuropathological processes, which could lead towards a clinical presentation of posterior cortical atrophy. Mutations in the presenilin 1 gene, affecting the function of gamma-secretase, are the most common genetic cause of familial, early-onset Alzheimer's disease. Here we present a patient with a clinical diagnosis of posterior cortical atrophy who harbors a novel Presenilin 1 mutation (I211M). In silico analysis predicts that the mutation could influence the interaction between presenilin 1 and presenilin1 enhancer-2 protein, a protein partner within the gamma-secretase complex. These findings along with published literature support the inclusion of posterior cortical atrophy on the Alzheimer's disease spectrum.
|
|
