Proteomic Characterization of Postmortem Amyloid Plaques Isolated by Laser Capture Microdissection

Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2004; 279(35):37061-8. DOI: 10.1074/jbc.M403672200
Source: PubMed


The presence of amyloid plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). We report here a comprehensive proteomic analysis of senile plaques from postmortem AD brain tissues. Senile plaques labeled with thioflavin-S were procured by laser capture microdissection, and their protein components were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified a total of 488 proteins co-isolated with the plaques, and we found multiple phosphorylation sites on the neurofilament intermediate chain, implicating the complexity and diversity of cellular processes involved in the plaque formation. More significantly, we identified 26 proteins enriched in the plaques of two AD cases by quantitative comparison with surrounding non-plaque tissues. The localization of several proteins in the plaques was further confirmed by the approach of immunohistochemistry. In addition to previously identified plaque constituents, we discovered novel association of dynein heavy chain with the plaques in human postmortem brain and in a double transgenic AD mouse model, suggesting that neuronal transport may play a role in neuritic degeneration. Overall, our results revealed for the first time the sub-proteome of amyloid plaques that is important for further studies on disease biomarker identification and molecular mechanisms of AD pathogenesis.

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    • "Postmortem AD brain samples have been extensively investigated by numerous proteomics platforms [33] [34] [35] [36] [37] [38], but systematic analysis of AD phosphoproteome has rarely reported [39]. "
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    ABSTRACT: Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a non-phosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 non-redundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain.This article is protected by copyright. All rights reserved
    Proteomics 10/2014; 15(2-3). DOI:10.1002/pmic.201400171 · 3.81 Impact Factor
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    • "For example, LCM has been combined with RNA isolation and transcriptome analysis to identify specific transcripts and components of the neuromuscular junction [5,6]. In combination with liquid chromatography tandem mass spectrometry (LC-MS/MS), LCM has been extensively used, amongst others applications, to purify and profile cancer cells (for a review see [7]), profile plaques in neurological disease [8-11] or elucidate the expression profile of inclusion bodies within muscle fibres [12]. In the experimental process of an expression profiling, LCM is often the limiting step, given the length of time required to recover a small amount of material. "
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    ABSTRACT: The myotendinous junction is a specialized structure of the muscle fibre enriched in mechanosensing complexes, including costameric proteins and core elements of the z-disc. Here, laser capture microdissection was applied to purify membrane regions from the myotendinous junctions of mouse skeletal muscles, which were then processed for proteomic analysis. Sarcolemma sections from the longitudinal axis of the muscle fibre were used as control for the specificity of the junctional preparation. Gene ontology term analysis of the combined lists indicated a statistically significant enrichment in membrane-associated proteins. The myotendinous junction preparation contained previously uncharacterized proteins, a number of z-disc costameric ligands (e.g., actinins, capZ, αB cristallin, filamin C, cypher, calsarcin, desmin, FHL1, telethonin, nebulin, titin and an enigma-like protein) and other proposed players of sarcomeric stretch sensing and signalling, such as myotilin and the three myomesin homologs. A subset were confirmed by immunofluorescence analysis as enriched at the myotendinous junction, suggesting that laser capture microdissection from muscle sections is a valid approach to identify novel myotendinous junction players potentially involved in mechanotransduction pathways.
    Proteome Science 05/2014; 12(25). DOI:10.1186/1477-5956-12-25 · 1.73 Impact Factor
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    • "abundantly studied (Liao, et al., 2004). A line of evidence indicates that lipids could play a major role in AD physiopathology (Basak, et al., 2012; Hashimoto, et al., 2012; Wolf, et al., 2004). "
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    ABSTRACT: The senile plaque is a hallmark lesion of Alzheimer disease (AD). We compared, without a priori, the lipidome of the senile plaques and of the adjacent plaque-free neuropil. The analysis by liquid chromatography coupled with electrospray ionization mass spectrometry revealed that laser microdissected senile plaques were enriched in saturated ceramides Cer(d18:1/18:0) and Cer(d18:1/20:0) by 33 and 78 % respectively with respect to the surrounding neuropil. This accumulation of ceramides was not explained by their affinity for Aβ deposits: no interaction between ceramide-liposomes and Aβ fibrils was observed in vitro by surface plasmon resonance and fluorescent ceramide-liposomes showed no affinity for the senile plaques in AD brain tissue. Accumulation of ceramides could be, at least partially, the result of a local production by acid and neutral sphingomyelinase that we found to be present in the corona of 78 % and 82 % of the senile plaque.
    Neurobiology of Disease 01/2014; 65. DOI:10.1016/j.nbd.2014.01.010 · 5.08 Impact Factor
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