| 1. |
- Andreasson, U., et al.
(författare)
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An enzyme activity as a potential biomarker for Alzheimer's disease.
- 2010
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Ingår i: Alzheimer's & Dementia. - : Wiley. - 1552-5279 .- 1552-5260. ; 6:4, s. 497-498
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Konferensbidrag (refereegranskat)abstract
- Background: Six different N-terminal amyloid precursor protein (APP) fragments, with molecular weight ∼12 kDa, have previously been identified in human cerebrospinal fluid (CSF). In a pilot study, both the sum of their concentrations, measured by western blot, and the relative abundance pattern, measured by mass spectrometry, were different in Alzheimer's disease (AD) patients compared to healthy controls. To test if these differences were also reflected in protease activities that possibly give rise to the ∼12 kDa fragments an enzymatic assay was developed and the activity in CSF was investigated for its potential as a biomarker for AD. Methods: The substrate in the protease activity assay was a custom made fluorochrome/quencher labeled peptide that covers the cleavage sites in APP (APP118-APP127) corresponding to the C-termini of the six ∼12 kDa APP fragments. The activity was measured in CSF from 55 AD patients and 17 controls. Results: There was a significant increase in the protease activity in CSF from AD patients compared to the controls (p = 0.001). This is in line with previous results which indicate that the sum of the ∼12 kDa fragments are elevated in AD. Results from inhibition studies strongly suggests that the enzyme responsible for the cleavage of the substrate is an aspartic protease since a sub nM IC50 value was recorded for Pepstatin A while no inhibition was observed for the cysteine protease specific inhibitor E64 at concentrations up to100 nM. Conclusions: There exists an enzymatic activity in CSF capable of cleaving a peptide substrate that spans a portion, close to the N-terminal, of APP. In a pilot study the activity is increased in AD patients compared to controls suggesting that it can be used as a biomarker.
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| 2. |
- Krastins, B., et al.
(författare)
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Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum
- 2013
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Ingår i: Clinical Biochemistry. - : Elsevier BV. - 0009-9120. ; 46:6, s. 399-410
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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.
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| 3. |
- Mattsson, Niklas, 1979, et al.
(författare)
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Reference measurement procedures for Alzheimer's disease cerebrospinal fluid biomarkers: definitions and approaches with focus on amyloid β42.
- 2012
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Ingår i: Biomarkers in medicine. - : Future Medicine Ltd. - 1752-0371 .- 1752-0363. ; 6:4, s. 409-17
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Tidskriftsartikel (refereegranskat)abstract
- Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid β42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid β42.
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