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Cloud-point extraction and delipidation of porcine brain proteins in combination with bottom-up mass spectrometry approaches for proteome analysis
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
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2010 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 9, no 8, 3903-3911 p.Article in journal (Refereed) Published
Abstract [en]

In this study, temperature-induced phase fractionation also known as cloud-point extraction (CPE) with the nonionic surfactant Triton X-114 was used to simultaneously extract hydrophobic and hydrophilic proteins from porcine brain tissue. Various protein precipitation/delipidation procedures were investigated to efficiently remove lipids and detergents while retaining maximum protein recoveries. The best performing delipidation method was then used in combination with CPE to compare three different mass spectrometry (MS) based "bottom-up" proteomic approaches for protein analysis of the porcine brain. In the first approach, the intact proteins were initially separated by one-dimensional (1D) gel electrophoresis. The excised protein bands were digested with trypsin, and the peptides were separated by reversed phase nanoliquid chromatography (RP-nanoLC) followed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) analysis. The other bottom-up proteomic approaches were based on first enzymatical digestion of the proteins followed by RP-nanoLC separation in combination with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) or on the combination of in-solution isoelectric focusing (IEF) with ESI-nanoLC-MS/MS of the IEF separated peptides. In total, we found and unambiguously identified 331 unique proteins. The overlap between different techniques was about 10%, showing that the use of multiple proteomic approaches is beneficial to yield a better coverage of the proteome. Furthermore, the overlap between the CPE extracted hydrophilic and hydrophobic proteins was rather small (9-16%), indicating an efficient sample preparation technique to extract and separate hydrophilic and hydrophobic proteins from brain tissue. The percentage of identified membrane proteins was 27%, which is in accordance to the fact that about one-third of all genes in various organisms encode for this class of proteins. The results indicate that cloud point extraction is a promising sample preparation tool, which allows simultaneous in depth studies of brain derived membrane proteins as well as hydrophilic proteins. This technique can be very useful when studying human central nervous system (CNS) tissue or animal models of neurological diseases.

Place, publisher, year, edition, pages
ACS , 2010. Vol. 9, no 8, 3903-3911 p.
Keyword [en]
cloud-point extraction (CPE), delipidation, central nervous system (CNS), brain, bottom-up proteomics, membrane proteins (MPs), mass spectrometry (MS)
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-130276DOI: 10.1021/pr100116kISI: 000280583700014PubMedID: 20586484OAI: oai:DiVA.org:uu-130276DiVA: diva2:349190
Available from: 2010-09-08 Created: 2010-09-06 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Advances for Biomarker Discovery in Neuroproteomics using Mass Spectrometry: From Method Development to Clinical Application
Open this publication in new window or tab >>Advances for Biomarker Discovery in Neuroproteomics using Mass Spectrometry: From Method Development to Clinical Application
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins offer a prominent group of compounds which may be ubiquitously affected in disease and used as biomarkers for early diagnosis, assessing treatment or drug development. Clinical proteomics aim to screen for protein biomarkers by a comprehensive analysis of all proteins expressed in a biological matrix during a certain pathology. Characterization of thousands of proteins in a complex biological matrix is from an analytical point of view a challenging task. Hence, sophisticated methods that are sensitive, specific and robust in a high-throughput manner are required. Mass spectrometry (MS) is able to perform this to a wide extent is.

A prominent source for finding protein biomarkers related to neurological diseases is the central nervous system (CNS) due to close proximity of the pathogenesis. Neuroproteomic analysis of CNS tissue samples is thus likely to reveal novel biomarkers. Cerebrospinal fluid (CSF) bathes the entire CNS and offers a good balance between clinical implementation and usefulness. Both matrices put further requirements on the methodology due to a high dynamic range, low protein concentration and limited sample amount.

The central objective of this thesis was to develop, assess and utilize analytical methods to be used in combination with MS to enable protein biomarker discovery in the CNS. The use of hexapeptide ligand libraries was exemplified on CSF from patients with traumatic brain injury and demonstrated the ability to compress the dynamic range to enable protein profiling in the order of mg/mL to pg/mL. Further, a method based on cloud-point extraction was developed for simultaneous enrichment and fractionation of hydrophobic/hydrophilic proteins in brain tissue. Comparison between label and label-free MS based strategies were carried out, mimicking the true conditions with a few differentially expressed proteins and a bulk of proteins occurring in unchanged ratio. Finally, a clinical application was carried out to explore the molecular mechanism underlying the analgesic effect of spinal cord stimulation (SCS) in patients with neuropathic pain. The CSF concentration of Lynx1 was found to increase upon SCS. Lynx1, acting as a specific modulator of the cholinergic system in the CNS, may act as a potential important molecular explanation of SCS-induced analgesia.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 966
Keyword
Mass Spectrometry, Biomarker, Proteomics, Central Nervous System, Cerebrospinal Fluid, Traumatic Brain Injury, Cloud-Point Extraction, Neuroproteomics, Relative Quantification, Spinal Cord Stimulation, Neuropathic Pain
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-180109 (URN)978-91-554-8457-6 (ISBN)
Public defence
2012-10-18, Biomedicinskt Centrum, B42, Husargatan 3, Uppsala, 10:15 (English)
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Available from: 2012-09-26 Created: 2012-08-29 Last updated: 2013-01-23Bibliographically approved

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Shevchenko, GannaSjödin, Marcus O.D.Malmström, DavidWetterhall, MagnusBergquist, Jonas

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