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Micellar extraction possesses a new advantage for the analysis of Alzheimer's disease brain proteome
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
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2015 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 407, no 4, 1041-1057 p.Article in journal (Refereed) Published
Abstract [en]

Integral membrane proteins (MPs), such as transporters, receptors, and ion channels, are of great interest because of their participation in various vital cellular functions including cell-cell interactions, ion transport, and signal transduction. However, studies of MPs are complicated because of their hydrophobic nature, heterogeneity, and low abundance. Cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was performed to simultaneously extract and phase separate hydrophobic and hydrophilic proteins from Alzheimer's disease (AD) and unaffected control brain tissue. Quantitative proteomics analysis of temporal neocortex samples of AD patients and controls was performed using a shotgun approach based on stable isotope dimethyl labeling (DML) quantification technique followed by nanoLC-MS/MS analysis. A total of 1096 unique proteins were identified and quantified, with 40.3 % (211/524) predicted as integral MPs with at least one transmembrane domain (TMD) found in the detergent phase, and 10 % (80/798) in the detergent-depleted phase. Among these, 62 proteins were shown to be significantly altered (p-value < 0.05), in AD versus control samples. In the detergent fraction, we found 10 hydrophobic transmembrane proteins containing up to 14 putative TMDs that were significantly up- or down-regulated in AD compared with control brains. Changes in four of these proteins, alpha-enolase (ENOA), lysosome-associated membrane glycoprotein 1 (LAMP1), 14-3-3 protein gamma (1433G), and sarcoplasmic/endoplasmic reticulum calcium ATPase2 (AT2A2) were validated by immunoblotting. Our results emphasize that separating hydrophobic MPs in CPE contributes to an increased understanding of the underlying molecular mechanisms in AD. Such knowledge can become useful for the development of novel disease biomarkers.

Place, publisher, year, edition, pages
2015. Vol. 407, no 4, 1041-1057 p.
Keyword [en]
Alzheimer's disease (AD), Cloud point extraction (CPE), Membrane proteins (MPs), Dimethyl labeling quantitative proteomics, Brain tissue
National Category
Geriatrics Analytical Chemistry
URN: urn:nbn:se:uu:diva-246344DOI: 10.1007/s00216-014-8320-8ISI: 000348436100002PubMedID: 25416231OAI: oai:DiVA.org:uu-246344DiVA: diva2:794057
Available from: 2015-03-10 Created: 2015-03-05 Last updated: 2016-04-04Bibliographically approved
In thesis
1. Mass Spectrometry-based Neuroproteomics: Deciphering the Human Brain Proteome
Open this publication in new window or tab >>Mass Spectrometry-based Neuroproteomics: Deciphering the Human Brain Proteome
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mammalian brain is challenging to study due to its heterogeneity and complexity. However, recent advances in molecular imaging, genomics and proteomics have contributed significantly to achieve insights into molecular basis of brain function and pathogenesis of neurological disorders. Efficient sample preparation is an integral part of a successful mass spectrometry (MS)-based proteomics. Apart from the identification, quantification of proteins is needed to investigate the alterations between proteome profiles from different sample sets. Therefore, this thesis investigates optimizing and application of the MS compatible sample preparation techniques for the identification and quantification of proteins from brain tissue.

The central objective of this thesis was (i) to improve the extraction of proteins as well as membrane proteins (MPs) from the brain tissue and (ii) to apply the optimized method along with the stable isotope dimethyl labeling (DML) and label-free (LF) MS approaches for the relative quantification of the brain proteome profiles during neurological conditions such as Alzheimer’s disease (AD) and traumatic brain injury (TBI).  First study described in this thesis is focused on the qualitative aspects for the brain tissue sample preparation. The optimized extraction buffers from first study containing n-octyl-β-glucopyranside or triton X-114 were used in the further quantitative studies to extract the proteins from patient (AD or TBI) and control human brain samples. Triton X-114 has additional advantage of separating MPs into a micellar phase. Therefore we also investigated the possibility to apply this in combination with DML quantitation approach for enrichment of low abundant MPs from AD brains.

AD and TBI causes severe socio-economic burden on the society and therefore there is a need to develop diagnostic markers to detect the early changes in the pathology of the disease. Analytical tools and techniques applied and discussed in this thesis for neuroproteomics applications proved to be powerful and reliable for analyzing complex biological samples to generate high-throughput screening and unbiased identification and quantitation of disease-specific proteins that are of great importance in understanding the disease pathology. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 70 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1347
Brain, Proteomics, Mass spectrometry, Alzheimer's disease, Traumatic brain injury, Membrane proteins, Sample preparation
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
urn:nbn:se:uu:diva-277613 (URN)978-91-554-9485-8 (ISBN)
Public defence
2016-04-08, B42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2016-03-18 Created: 2016-02-22 Last updated: 2016-04-04

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Musunuri, SravaniKultima, KimIngelsson, MartinLannfelt, LarsBergquist, JonasShevchenko, Ganna
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