Investigation of laser-microdissected inclusion bodies.
We established a novel combinatorial method of laser microdissection system and immunoblot analysis in combination with a novel unfolding chaperone (oligomeric Aip2p/Dld2p) that enables us to examine the molecular profile of proteins in the microscopic regions of interest. As a model system for analyzing inclusion bodies associated with various diseases such as Alzheimer's disease, Parkinson's disease, and prion diseases including bovine spongiform encephalopathy (BSE), we applied this novel method to examine brain samples of patients with Pick's disease, a type of progressive presenile dementia with intraneuronal lesions denoted as Pick bodies (PBs) whose major structural components are tau proteins. After boiling in Laemmli's sample buffer according to the established immunoblotting procedures, 500-2000 PBs were initially applied onto SDS-PAGE gels; however, only faint signals were obtained. Remarkably, only one Pick body was sufficient to illustrate an immunoblot signal; this indicates that pretreatment with oligomeric Aip2p/Dld2p enhances the immunoblot sensitivity by more than a 100-fold. This unprecedented property of laser microdissection combined with oligomeric Aip2p/Dld2p may have further potential applications. For example, a number of proteomic strategies for such inclusion bodies depend on liquid chromatography-tandem mass spectrometry (LC-MS/MS); however, sample preparation methods typically involve the use of detergents and chaotropic agents that often interfere with chromatographic separation and/or electrospray ionization. However, the use of oligomeric Aip2p/Dld2p would not interfere with the LC-MS/MS procedures. Therefore, it might significantly facilitate nanoscale analysis, which is often hindered by the aggregation property of the target proteins present under various analytical conditions, particularly, when the sample protein is present in minor quantities.
Hachiya
Naomi S
NS
Department of Neurophysiology, Tokyo Medical University 6-1-1 Shinjuku, Shinjuku-ku Tokyo 160-8402, Japan.
Kaneko
Kiyotoshi
K
D000368:Aged; D001921:Brain / Q000378:metabolism; D005260:Female; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000648:ultrastructure; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D042844:L-Lactate Dehydrogenase (Cytochrome); D007834:Lasers; D008297:Male; D042282:Microdissection / Q000379:methods*; D008875:Middle Aged; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000302:isolation & purification / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D012995:Solubility / Q000187:drug effects; D016875:tau Proteins / Q000302:isolation & purification / Q000378:metabolism
D020033:Protein Isoforms; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D016875:tau Proteins; C025231:sarkosyl; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); C487563:DLD2 protein, S cerevisiae; D012521:Sarcosine