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.
This work was funded by the Swedish Research Council (VR 621-2010-4206), Foundation for Strategic Research (SBE13-0115), Stockholm County Council through an ALF grant, funds from Karolinska University Hospital and Karolinska Institutet, Swedish Brain Foundation (Hjärnfonden) and the EMRP Projects IND15 “SurfChem” and HLT04 “BioSurf.” The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union. The authors would also like to acknowledge Sabina Burazerovic for all help using the gel filtration and Alina Codita for preparing and supplying the mouse brain tissue samples.
I. INTRODUCTION II. EXPERIMENTAL SECTION A. Preparation and characterization of immunoliposomes B. QCM-D analysis C. Preparation of tissue sections from mouse brains D. Immunohistochemistry and fluorescence microscopy E. ToF-SIMS analysis III. RESULTS AND DISCUSSION A. Characterization of specific binding of immunoliposomes to a model surface using QCM-D B. Immunoliposome binding and localization of Aβ in tissue sections with ToF-SIMS and fluorescence microscopy C. Simultaneous imaging of Aβ and lipids with ToF-SIMS and investigation of the lipid distribution IV. SUMMARY AND CONCLUSIONS