Detection of Lipids and Proteins on Biological Surfaces using Imaging Mass Spectrometry

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

The impact of detergents on the tissue decellularization process: a ToF-SIMS study

Biologic scaffolds are derived from mammalian tissues, which must be decellularized to remove cellular antigens that would otherwise incite an adverse immune response. Although widely used clinically, the optimum balance between cell removal and the disruption of matrix architecture and surface ligand landscape remains a considerable challenge. Here we describe the use of time of flight secondary ion mass spectroscopy (ToF-SIMS) to provide sensitive, molecular specific, localized analysis of detergent decellularized biologic scaffolds. We detected residual detergent fragments, specifically from Triton X-100, sodium deoxycholate and sodium dodecyl sulphate (SDS) in decellularized scaffolds; increased SDS concentrations from 0.1% to 1.0% increased both the intensity of SDS fragments and adverse cell outcomes. We also identified cellular remnants, by detecting phosphate and phosphocholine ions in PAA and CHAPS decellularized scaffolds. The present study demonstrates ToF-SIMS is not only a powerful tool for characterization of biologic scaffold surface molecular functionality, but also enables sensitive assessment of decellularization efficacy

Imaging of lipids and proteins in Alzheimer\u27s disease using Time-of-Flight Secondary Ion Mass Spectrometry

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

Detection of Lipids and Proteins on Biological Surfaces using Imaging Mass Spectrometry

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

Imaging of Amyloid-β in Alzheimer’s disease transgenic mouse brains with Time-of-Flight Secondary Ion Mass Spectrometry using Immunoliposomes

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\u27s 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

Imaging of Amyloid-β in Alzheimer’s disease transgenic mouse brains with Time-of-Flight Secondary Ion Mass Spectrometry using Immunoliposomes

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\u27s 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

Simultaneous imaging of amyloid-β and lipids in brain tissue using antibody-coupled liposomes and time-of-flight secondary ion mass spectrometry

The spatial localization of amyloid-β peptide deposits, the major component of senile plaques in Alzheimer\u27s 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. \ua9 2014 American Chemical Society

Simultaneous Imaging of Amyloid‑β and Lipids in Brain Tissue Using Antibody-Coupled Liposomes and Time-of-Flight Secondary Ion Mass Spectrometry

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