Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the central nervous system. Two pathological hallmarks in the brain of AD patients are neurofibrillary tangles and senile plaques. The plaques consist mainly of beta-amyloid (Abeta) peptides that are produced from the amyloid precursor protein (APP), by sequential cleavage by beta- and gamma-secretase. Most previous studies have been focused on the C-terminal fragments of APP, where the Abeta sequence is localized. The purpose of this study was to search for N-terminal fragments of APP in cerebrospinal fluid (CSF) using mass spectrometry (MS). By using immunoprecipitation (IP) combined with matrix-assisted laser desorption/ionization time-of-flight MS as well as nanoflow liquid chromatography coupled to high resolution tandem MS we were able to detect and identify six novel N-terminal APP fragments [APP((18-119)), APP((18-121)), APP((18-122)), APP((18-123)), APP((18-124)) and APP((18-126))], having molecular masses of approximately 12 kDa. The presence of these APP derivatives in CSF was also verified by Western blot analysis. Two pilot studies using either IP-MS or Western blot analysis indicated slightly elevated levels of N-terminal APP fragments in CSF from AD patients compared with controls, which are in need of replications in independent and larger patient materials.
"For example, APP is cleaved by a-or b-secretase to produce ectodomain fragments (sAPPa or sAPPb) that are secreted into the extracellular milieu (Nunan and Small, 2000). Several studies have shown that sAPPa and APP can be cleaved, both in vitro and in vivo, to yield smaller N-terminal fragments (Small et al., 1994; Caswell et al., 1999: Esh et al., 2005; Nikolaev et al., 2009; Portelius et al., 2010; Jefferson et al., 2011; Vella and Cappai, 2012). The level of APP N-terminal fragments has been reported to increase during development as cells differentiate into neurons (Vella and Cappai, 2012). "
"α-Syn from human brain tissue was biochemically fractionated in absence or presence of different detergents leading to separation of α-syn according to solubility. We have in this study applied a hybrid approach, where an affinity based purification is combined with two different top-down mass spectrometry approaches to analyze the selectively purified endogenous peptides and proteins [27–30]. An immunoprecipitation method was optimized to analyze α-syn in the brain tissue homogenates by matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOFMS). "
[Show abstract][Hide abstract] ABSTRACT: Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative diseases that are characterized by intra-neuronal inclusions of Lewy bodies in distinct brain regions. These inclusions consist mainly of aggregated α-synuclein (α-syn) protein. The present study used immunoprecipitation combined with nanoflow liquid chromatography (LC) coupled to high resolution electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) to determine known and novel isoforms of α-syn in brain tissue homogenates. N-terminally acetylated full-length α-syn (Ac-α-syn₁₋₁₄₀) and two N-terminally acetylated C-terminally truncated forms of α-syn (Ac-α-syn₁₋₁₃₉ and Ac-α-syn₁₋₁₀₃) were found. The different forms of α-syn were further studied by Western blotting in brain tissue homogenates from the temporal cortex Brodmann area 36 (BA36) and the dorsolateral prefrontal cortex BA9 derived from controls, patients with DLB and PD with dementia (PDD). Quantification of α-syn in each brain tissue fraction was performed using a novel enzyme-linked immunosorbent assay (ELISA).
Neurochemical Research 06/2011; 36(11):2029-42. DOI:10.1007/s11064-011-0527-x · 2.59 Impact Factor
"In a different study which was supported by cNEUPRO, six novel N-terminal APP-fragments with molecular masses of approximately 12 kDa and starting at amino acid 18 of the APP sequence were detected in CSF by mass spectrometry. In a subsequent small pilot study, six of six AD patients and five of five controls could be classified correctly by the combined evaluation of five of the six fragments . Additionally, Immuno-MS analysis of CSF has led to the detection of eleven novel APP fragments, which begin N-terminally to the β-secretase cleavage site, and end one amino acid before the proposed α-secretase cleavage site (APP/Aβ peptides) . "
[Show abstract][Hide abstract] ABSTRACT: "clinical NEUroPROteomics of neurodegenerative diseases" (cNEUPRO) is a Specific Targeted Research Project (STREP) within the sixth framework program of the European Commission dedicated to the search for novel biomarker candidates for Alzheimer's disease and other neurodegenerative diseases. The ultimate goal of cNEUPRO is to identify one or more valid biomarker(s) in blood and CSF applicable to support the early and differential diagnosis of dementia disorders. The consortium covers all steps required for the discovery of novel biomarker candidates such as acquisition of high quality CSF and blood samples from relevant patient groups and controls, analysis of body fluids by various methods, and finally assay development and assay validation. Here we report the standardized procedures for diagnosis and preanalytical sample-handling within the project, as well as the status of the ongoing research activities and some first results.
International Journal of Alzheimer's Disease 09/2010; 2010. DOI:10.4061/2010/548145
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