RESUMO
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) provides a unique in situ chemical profile that can include drugs, nucleic acids, metabolites, lipids, and proteins. MSI of individual cells (of a known cell type) affords a unique insight into normal and disease-related processes and is a prerequisite for combining the results of MSI and other single-cell modalities (e.g. mass cytometry and next-generation sequencing). Technological barriers have prevented the high-throughput assignment of MSI spectra from solid tissue preparations to their cell type. These barriers include obtaining a suitable cell-identifying image (e.g. immunohistochemistry) and obtaining sufficiently accurate registration of the cell-identifying and MALDI-MS images. This study introduces a technique that overcame these barriers by assigning cell type directly from mass spectra. We hypothesized that, in MSI from mice with a defined fluorescent protein expression pattern, the fluorescent protein's molecular ion could be used to identify cell cohorts. A method was developed for the purification of enhanced yellow fluorescent protein (EYFP) from mice. To determine EYFP's molecular mass for MSI studies, we performed intact mass analysis and characterized the protein's primary structure and post-translational modifications through various techniques. MALDI-MSI methods were developed to enhance the detection of EYFP in situ, and by extraction of EYFP's molecular ion from MALDI-MS images, automated, whole-image assignment of cell cohorts was achieved. This method was validated using a well-characterized mouse line that expresses EYFP in motor and sensory neurons and should be applicable to hundreds of commercially available mice (and other animal) strains comprising a multitude of cell-specific fluorescent labels.
Assuntos
Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Imagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Camundongos , Peso Molecular , Neurônios/metabolismoRESUMO
Imaging mass spectrometry (IMS) is a novel quantitative technique used to investigative diverse biomolecules in tissue sections. Specifically, IMS uses analytical separation of mass spectrometry to determine the spatial distribution of certain lipids and/or proteins located directly on biological sections from a single tissue sample. Typically, IMS is combined with histological analysis to reveal additional distribution details of characterized biomolecules including cell type and/or subcellular localization. In this chapter, we describe the use of Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-Of-Flight/Time-Of-Flight (TOF/TOF) to analyze various cholesterol and phosphatidylcholine species in atherosclerotic plaque of murine heart aortic valves. In particular, we detail animals used, tissue collection, preparation, matrix application, spectra acquisition for generating a color-coded image based on IMS spectral characteristics.
Assuntos
Espectrometria de Massas/métodos , Metabolômica/métodos , Animais , Apolipoproteínas E/genética , Biomarcadores , Feminino , Camundongos , Camundongos Knockout , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The resolution of MALDI MS imaging is limited by the displacement of analytes during matrix deposition or by laser focal diameter. Here we present three methods that minimize the displacement of analytes during matrix deposition, including a method where image resolution is not limited by the laser focal diameter. The first method, matrix solution fixation, simultaneously fixes tissue while depositing matrix and is optimal for analyzing proteins and for applications requiring a fast preparation time. This method is characterized by compatibility with histology methods and laser focal diameter-limited resolution. The second method, a sensor controlled aerosol, is characterized by aerosol droplet size-limited resolution and is optimal for small molecules, including lipids, peptides, and drug-like molecules. The third method, microinjection with matrix, selectively deposits matrix upon cells of interest, offers cellular resolution and is compatible with most analytes. A flow chart summarizing methods is provided so that users may design a tissue preparation strategy based upon their resources and experimental goals.