Genetically Encoded Fluorescent Proteins Enable High-Throughput Assignment of Cell Cohorts Directly from MALDI-MS Images

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March 16, 2019

Authors

Nicholas D. Schmitt¹, Catherine M. Rawlins¹, Elizabeth C. Randall², Xianzhe Wang¹, Antonius Koller¹, Jared R. Auclair^1,3, Jane-Marie Kowalski⁴, Paul J. Kowalski⁴, Ed Luther⁵, Alexander R. Ivanov¹, Nathalie Y.R. Agar^2,6, and Jeffrey N. Agar^1,5

Organizations

Department of Chemistry and Chemical Biology and Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, Massachusetts 02115
Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115
Biopharmaceutical Analysis Training laboratory (BATL), Northeastern University Innovation Campus, Burlington, Massachusetts, 01803
Bruker Daltonics, 40 Manning Road, Billerica, Massachusetts 01821
Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115
Department of Neurosurgery, Brigham and Women’s Hospital, Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Abstract

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.

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Discussion

This article makes reference to CovalX technolgoies discussing the sensitivity of typical mass spectrometers decreasing as the protein size increases. Products like the CovalX HM1 detection system are suggested as solutions this problem.