| 1. |
- Carlred, Louise M, 1985
(författare)
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Detection of Lipids and Proteins on Biological Surfaces using Imaging Mass Spectrometry
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a technique that can be used for imaging the spatial distribution of many different molecules at the same time. It is very sensitive for detection of small biomolecules, such as lipids, whereas larger biomolecules, such as peptides and proteins, cannot be detected as intact entities due to fragmentation. In this work, we have explored an alternative approach for detection of peptides and proteins with ToF-SIMS, using liposomes for labeling the target of interest. In this way, both lipids and proteins can be imaged at the same time, which opens up for the opportunity to investigate lipid-protein interactions. The method has been applied for detection of biomolecules on two different biological surfaces; (1) a model surface containing controlled concentrations of target biomolecules bound to the substrate and (2) brain tissue sections from Alzheimer’s disease transgenic mice. Other techniques, such as fluorescence microscopy and quartz crystal microbalance with dissipation monitoring (QCM-D), have also been used for the characterization of liposomes binding to the surface. Another imaging mass spectrometry technique, matrix-assisted laser desorption/ionization (MALDI), was also employed on mouse brain tissue sections for detection and investigation of amyloid-β deposits, a peptide associated with Alzheimer’s disease. This thesis thus shows how different techniques can be combined for investigation of biomolecules on complex biological surfaces, in order to potentially provide new information about the mechanism of neurodegeneration in Alzheimer’s disease.
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| 2. |
- Carlred, Louise M, 1985, et al.
(författare)
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Imaging of Amyloid-β in Alzheimer’s disease transgenic mouse brains with Time-of-Flight Secondary Ion Mass Spectrometry using Immunoliposomes
- 2016
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Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 11:2, s. 1-11
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Tidskriftsartikel (refereegranskat)abstract
- Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been proven to successfully image different kinds of molecules, especially a variety of lipids, in biological samples. Proteins, however, are difficult to detect as specific entities with this method due to extensive fragmentation. To circumvent this issue, the authors present in this work a method developed for detection of proteins using antibody-conjugated liposomes, so called immunoliposomes, which are able to bind to the specific protein of interest. In combination with the capability of ToF-SIMS to detect native lipids in tissue samples, this method opens up the opportunity to analyze many different biomolecules, both lipids and proteins, at the same time, with high spatial resolution. The method has been applied to detect and image the distribution of amyloid-β (Aβ), a biologically relevant peptide in Alzheimer's disease (AD), in transgenic mouse braintissue. To ensure specific binding, the immunoliposome binding was verified on a model surface using quartz crystal microbalance with dissipation monitoring. The immunoliposome binding was also investigated on tissue sections with fluorescence microscopy, and compared with conventional immunohistochemistry using primary and secondary antibodies, demonstrating specific binding to Aβ. Using ToF-SIMS imaging, several endogenous lipids, such as cholesterol and sulfatides, were also detected in parallel with the immunoliposome-labeled Aβ deposits, which is an advantage compared to fluorescence microscopy. This method can thus potentially provide further information about lipid–protein interactions, which is important to understand the mechanisms of neurodegeneration in AD.
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| 3. |
- Carlred, Louise M, 1985
(författare)
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Imaging of lipids and proteins in Alzheimer's disease using Time-of-Flight Secondary Ion Mass Spectrometry
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the formation of senile plaques. These plaques, which consist of aggregations of a peptide called amyloid-β, are deposited in-between the nerve cells in the brain, where they disrupt the signaling processes. The reason for the generation of these plaques is not completely known, but one has found that the regulation of lipids, such as cholesterol, in the cell membrane is one of many factors involved in the process.One method used to study the generation of AD is imaging of brain tissue samples with fluorescence microscopy. To be able to study individual types of molecules in the tissue, immunohistochemistry is often applied, in which antibodies are used to target the molecule of interest. In this way, several different proteins can be visualized simultaneously, while lipids often remain unseen. Another method that can be used to image molecules in tissue samples, and especially lipids, is time-of-flight secondary ion mass spectrometry (ToF-SIMS). However, this method cannot detect intact molecules over ~2 kDa, thereby excluding most peptides and proteins from being identified.In this work, the capability of ToF-SIMS to detect lipids is utilized for targeting proteins in tissue samples using antibody-coupled lipid vesicles, so called liposomes. The antibody-coupled liposomes were specifically bound to amyloid-β deposits in transgenic AD mouse brains, enabling ToF-SIMS imaging of both amyloid-β and, at the same time, surrounding lipids, such as cholesterol, in the tissue. The specificity of the liposome binding was investigated by analyzing their interaction with a model surface using quartz crystal microbalance with dissipation monitoring (QCM-D). Furthermore, the binding of the antibody-coupled liposomes to amyloid-β deposits in tissue sections was analyzed with fluorescence microscopy, confirming specific binding. To unravel possible artifacts in the tissue sample due to the demanding sample preparation required for ToF-SIMS imaging, the effects of the tissue preparation protocol were using ToF-SIMS and scanning electron microcopy (SEM), revealing no severe spatial redistribution of the native lipids or any major disruption of the surface morphology. This method may thus provide an important complement to traditional tissue imaging approaches for the investigation of the interaction between lipids and proteins, which may result in important clues about the generation of different diseases, such as AD.
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| 4. |
- Carlred, Louise M, 1985, et al.
(författare)
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Probing Amyloid-β Pathology in transgenic Alzheimer's disease (tgArcSwe) mice using MALDI Imaging Mass Spectrometry
- 2016
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Ingår i: Journal of neurochemistry. - : Wiley. - 1471-4159 .- 0022-3042. ; 138:3, s. 469-478
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Tidskriftsartikel (refereegranskat)abstract
- The pathological mechanisms underlying Alzheimer's disease (AD) are still not understood. The disease pathology is characterized by accumulation and aggregation of amyloid-β (Aβ) peptides into extracellular plaques, however the factors that promote neurotoxic Aβ aggregation remain elusive. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides and proteins in biological tissues. In the present study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) based imaging was used to study Aβ deposition in transgenic mouse brain tissue and to elucidate the plaque associated chemical microenvironment. The imaging experiments were performed in brain sections of transgenic Alzheimer's disease mice carrying the Arctic and Swedish mutation of amyloid-beta precursor protein (tgArcSwe). Multivariate image analysis was used to interrogate the IMS data for identifying pathologically relevant, anatomical features based on their chemical identity. This include cortical and hippocampal Aβ deposits, whose amyloid peptide content was further verified using immunohistochemistry and laser micro dissection followed by MALDI MS analysis. Subsequent statistical analysis on spectral data of regions of interest (ROI) revealed brain region specific differences in Aβ peptide aggregation. Moreover, other plaque associated protein species were identified including macrophage migration inhibitory factor (MIF) suggesting neuroinflammatory processes and glial cell reactivity to be involved in AD pathology. The presented data further highlight the potential of IMS as powerful approach in neuropathology.
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| 5. |
- Carlred, Louise M, 1985, et al.
(författare)
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Simultaneous imaging of amyloid-β and lipids in brain tissue using antibody-coupled liposomes and time-of-flight secondary ion mass spectrometry
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 136:28, s. 9973-9981
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Tidskriftsartikel (refereegranskat)abstract
- The spatial localization of amyloid-β peptide deposits, the major component of senile plaques in Alzheimer's disease (AD), was mapped in transgenic AD mouse brains using time-of-flight secondary ion mass spectrometry (ToF-SIMS), simultaneously with several endogenous molecules that cannot be mapped using conventional immunohistochemistry imaging, including phospholipids, cholesterol and sulfatides. Whereas the endogenous lipids were detected directly, the amyloid-β deposits, which cannot be detected as intact entities with ToF-SIMS because of extensive ion-induced fragmentation, were identified by specific binding of deuterated liposomes to antibodies directed against amyloid-β. Comparative investigation of the amyloid-β deposits using conventional immunohistochemistry and fluorescence microscopy suggests similar sensitivity but a more surface-confined identification due to the shallow penetration depth of the ToF-SIMS signal. The recorded ToF-SIMS images thus display the localization of lipids and amyloid-β in a narrow (∼10 nm) two-dimensional plane at the tissue surface. As compared to a frozen nontreated tissue sample, the liposome preparation protocol generally increased the signal intensity of endogenous lipids, likely caused by matrix effects associated with the removal of salts, but no severe effects on the tissue integrity and the spatial distribution of lipids were observed with ToF-SIMS or scanning electron microscopy (SEM). This method may provide an important extension to conventional tissue imaging techniques to investigate the complex interplay of different kinds of molecules in neurodegenerative diseases, in the same specimen. However, limitations in target accessibility of the liposomes as well as unspecific binding need further consideration. © 2014 American Chemical Society.
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| 6. |
- Sjövall, Peter, 1961, et al.
(författare)
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Liposome binding for multiplexed biomolecule detection and imaging using ToF-SIMS
- 2014
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Ingår i: Surface and Interface Analysis. - : Wiley. - 1096-9918 .- 0142-2421. ; 46:10-11, s. 707-711
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Tidskriftsartikel (refereegranskat)abstract
- A novel approach for multiplexed biomolecule detection at surfaces, involving specific binding of liposomes and subsequent analysis by time-of-flight secondary ion mass spectrometry (ToF-SIMS), was evaluated with respect to its capability for quantitative analysis of biomolecule surface concentrations. The specific binding of liposomes to a poly(L-lysine)-g-poly (ethylene glycol) (PLL-g-PEG) surface, using the biotin-avidin coupling chemistry, was characterized by quartz crystal microbalance with dissipation monitoring (QCM-D), fluorescence microscopy and ToF-SIMS. The ToF-SIMS results showed a linearly increasing signal from the liposomes up to a saturation coverage corresponding to a full liposome layer on the surface, in close agreement with fluorescence microscopy analysis of the same samples, strongly supporting the potential of the liposome-based approach for quantitative biomolecule detection. However, the multiplexing capability and issues on nonspecific binding need further studies. Furthermore, an improved method for the preparation of lipid bilayer samples for ToF-SIMS analysis is presented, demonstrating the imaging of individual 210-nm diameter liposomes adsorbed on a SiO2 surface.
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