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Mass Spectrometry-based Neuroproteomics: Deciphering the Human Brain Proteome
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University.
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1347
Keyword [en]
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
Identifiers
URN: urn:nbn:se:uu:diva-277613ISBN: 978-91-554-9485-8 (print)OAI: oai:DiVA.org:uu-277613DiVA: diva2:905205
Public defence
2016-04-08, B42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2016-03-18 Created: 2016-02-22 Last updated: 2016-04-04
List of papers
1. Comparison of Extraction Methods for the Comprehensive Analysis of Mouse Brain Proteome using Shotgun-based Mass Spectrometry
Open this publication in new window or tab >>Comparison of Extraction Methods for the Comprehensive Analysis of Mouse Brain Proteome using Shotgun-based Mass Spectrometry
2012 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 4, 2441-2451 p.Article in journal (Refereed) Published
Abstract [en]

This study compares 16 different extraction methods for the comprehensive extraction of mouse brain proteome in combination with "shotgun"-based mass spectrometry (MS). Membrane proteins (MPs) are responsible for a large part of the regulatory functions of the cell and are therefore of great interest to extract and analyze. Sixteen protein extraction protocols were evaluated in regards to protein yield and number of identified proteins with emphasis on MPs. The extracted proteins were delipidated, on-filter digested, and analyzed by reversed phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQ: FT mass spectrometer. Detergent-based lysis buffers showed higher efficiencies and yields in the extraction of proteins from the brain tissue compared to solubilization with organic solvents or organic acids. The detergent octyl-beta-D-glucopyranoside gave the highest number of identified proteins (541) as well as numbers and percentages of identified MPs (29%). Detergent-based protocols are the best sample preparation tools for central nervous system (CNS) tissue and can readily be applied to screen for candidate biomarkers of neurological diseases.

Keyword
brain, proteomics, membrane proteins (MPs), transmembrane proteins (TMPs), mass spectrometry (MS), sample preparation, extraction
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-173655 (URN)10.1021/pr201169q (DOI)000302388100034 ()
Available from: 2012-05-02 Created: 2012-05-02 Last updated: 2017-12-07Bibliographically approved
2. Quantification of the Brain Proteome in Alzheimer's Disease Using Multiplexed Mass Spectrometry
Open this publication in new window or tab >>Quantification of the Brain Proteome in Alzheimer's Disease Using Multiplexed Mass Spectrometry
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2014 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 4, 2056-2068 p.Article in journal (Refereed) Published
Abstract [en]

We have compared the brain proteome in the temporal neocortex between Alzheimer's disease (AD) patients and non-AD individuals by using shotgun mass spectrometry based on a stable isotope dimethyl labeling. A total of 827 unique proteins were identified and quantitated. Of these, 227 proteins were found in at least 9 out of 10 AD/control pairs and were further subjected to statistical analysis. A total of 69 proteins showed different levels (p-value < 0.05) in AD versus control brain samples. Of these proteins, 37 were increased and 32 were decreased as compared to the non-AD subjects. Twenty-three proteins comprise novel proteins that have not previously been reported as related to AD, e.g., neuronal-specific septin-3, septin-2, septin-5, dihydropteridine reductase, and clathrin heavy chain 1. The proteins with altered levels in the AD brain represent a wide variety of pathways suggested to be involved in the disease pathogenesis, including energy metabolism, glycolysis, oxidative stress, apoptosis, signal transduction, and synaptic functioning. Apart from leading to new insights into the molecular mechanisms in AD, the findings provide us with possible novel candidates for future diagnostic and prognostic disease markers.

Keyword
Alzheimer's disease (AD), dimethyl labeling (DML), quantitative proteomics, mass spectrometry (MS), brain tissue
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-224578 (URN)10.1021/pr401202d (DOI)000334016400025 ()
Available from: 2014-05-19 Created: 2014-05-14 Last updated: 2017-12-05Bibliographically approved
3. Micellar extraction possesses a new advantage for the analysis of Alzheimer's disease brain proteome
Open this publication in new window or tab >>Micellar extraction possesses a new advantage for the analysis of Alzheimer's disease brain proteome
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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.

Keyword
Alzheimer's disease (AD), Cloud point extraction (CPE), Membrane proteins (MPs), Dimethyl labeling quantitative proteomics, Brain tissue
National Category
Geriatrics Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-246344 (URN)10.1007/s00216-014-8320-8 (DOI)000348436100002 ()25416231 (PubMedID)
Available from: 2015-03-10 Created: 2015-03-05 Last updated: 2017-12-04Bibliographically approved
4. Increased levels of extracellular microvesicle markers and decreased levels of endocytic/exocytic proteins in the Alzheimer’s disease brain
Open this publication in new window or tab >>Increased levels of extracellular microvesicle markers and decreased levels of endocytic/exocytic proteins in the Alzheimer’s disease brain
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2016 (English)In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 54, no 4, 71 p.1671-1686 p.Article in journal (Refereed) Published
Abstract [en]

Background: Alzheimer's disease (AD) is a chronic neurodegenerative disorder accounting for more than 50% of all dementia cases. AD neuropathology is characterized by the formation of extracellular plaques and intracellular neurofibrillary tangles consisting of aggregated amyloid-beta and tau, respectively. The disease mechanism has only been partially elucidated and is believed to also involve many other proteins.

Objective: This study intended to perform a proteomic profiling of post mortem AD brains and compare it with control brains as well as brains from other neurological diseases to gain insight into the disease pathology.

Methods: Here we used label-free shotgun mass spectrometry to analyze temporal neocortex samples from AD, other neurological disorders, and non-demented controls, in order to identify additional proteins that are altered in AD. The mass spectrometry results were verified by antibody suspension bead arrays.

Results: We found 50 proteins with altered levels between AD and control brains. The majority of these proteins were found at lower levels in AD. Pathway analyses revealed that several of the decreased proteins play a role in exocytic and endocytic pathways, whereas several of the increased proteins are related to extracellular vesicles. Using antibody-based analysis, we verified the mass spectrometry results for five representative proteins from this group of proteins (CD9, HSP72, PI42A, TALDO, and VAMP2) and GFAP, a marker for neuroinflammation.

Conclusions: Several proteins involved in exo-endocytic pathways and extracellular vesicle functions display altered levels in the AD brain. We hypothesize that such changes may result in disturbed cellular clearance and a perturbed cell-to-cell communication that may contribute to neuronal dysfunction and cell death in AD.

Publisher
71 p.
Keyword
Brain, Proteomics, Mass spectrometry, Alzheimer's disease
National Category
Analytical Chemistry Geriatrics Neurosciences
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-277617 (URN)10.3233/JAD-160271 (DOI)000386749900034 ()27636840 (PubMedID)
Funder
VINNOVALars Hierta Memorial FoundationSwedish Research Council, P29797-1; 621-2011-4423Knut and Alice Wallenberg FoundationStiftelsen Gamla Tjänarinnor
Available from: 2016-02-22 Created: 2016-02-22 Last updated: 2017-10-17Bibliographically approved
5. Differential proteomic analysis for the evaluation of protein expression in fresh human cortical brain biopsies
Open this publication in new window or tab >>Differential proteomic analysis for the evaluation of protein expression in fresh human cortical brain biopsies
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(English)Manuscript (preprint) (Other academic)
Keyword
Mass spectrometry, Neuroproteomics, Traumatic brain injury
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-277618 (URN)
Available from: 2016-02-22 Created: 2016-02-22 Last updated: 2016-04-04

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