Method Development for Improvement of Subcellular Resolution in Mass Spectrometry Imaging

Laser desorption/ionization mass spectrometry (LDI-MS) imaging has a much higher channel throughput than optical imaging and can detect known or unknown biomolecules. This unique technical feature of mass spectrometry imaging puts it in a pivotal position in medical discovery. However, the resolution of MS imaging is much lower than optical imaging, limiting its application in biomedical research. Therefore, the development of mass spectrometry screening of molecular composition at the cellular and subcellular resolution is the central goal of contemporary matrix-assistant laser desorption/ionization (MALDI)-MS imaging. High spatial resolution MS imaging can be achieved by improving the laser optical system of the mass spectrometer. State-of-art values on the spatial resolution of commercial MS imaging systems range from 5-20 μm, which is far below the need for subcellular resolution. Although sub-cellular resolution can be achieved through the development of more advanced mass spectrometry laser optical systems, the commercialization of instruments and their applications may take ten years later.

This project will develop a multiplex and subcellular resolution protein imaging method based on a commercially available imaging system by physically expanding the size of tissues. The strategy is based on expansion microscopy. As the gel expands, the tissue expands in an equal proportion. Then targeted proteins will incubate with photocleavable mass-tags labeled antibodies. The photocleavable linker is broken when the laser is excited, and MS will detect the dissociated and ionized mass tags. It is expected a 2.5 to 1μm resolution could be achieved using typical commercial instruments, which is a remarkable improvement from its original 10μm resolution. The technology to be developed is an independent sample pretreatment process so that it can be universally applied to a variety of MS platforms. It can proportionally improve the resolution of a wide range of MS imaging systems. Since the sample expansion and the mass spectrometer laser optical system are orthogonally independent, the combination of this method and the next-generation higher resolution mass spectrometry imaging system is expected to provide imaging resolution comparable to that of conventional optical microscopes. Compared with optical microscopes used for protein immunofluorescence imaging, this method can provide higher channel throughput. This research reduces the reliance on instruments and will have a broad and immediate boost to medical and biological research. This research also lays the foundation for the development of next- generation label-free expansion MS imaging strategies.