Fixing cells prior to XL-MS for improved sampling of the spatial proteome
1 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N-4N1, Canada
2 Department of Chemistry, University of Calgary, Calgary, Alberta T2N-4N1, Canada
Introduction: XL-MS can be used to map protein-protein interactions (PPIs) in situ, ideally capturing them in their native environment and reflecting the organizational properties of the proteome. However, the nature of the cellular medium hinders the efficiency of the crosslinking experiment and limits our ability to deeply sample PPI space. The dynamics of the proteome during long crosslinker incubation times promotes reagent scavenging and distortion of the spatial proteome. Ideally, the proteome should be stabilized prior to crosslinking. Fixation strategies have been used successfully in microscopy for many years. We present two concepts for integrating cell fixation with XL-MS, one based on cryogenic methods and the other on classical formalin fixation, both designed to improve the utility of in situ XL-MS.
Methods: Our apparatus for cryogenic crosslinking includes a spray-freezer of cell suspensions, a specially designed unit for desiccating cells with -80°C acetone, and an adapted autosampler/pump system for freeze-substituting protein-reactive compounds at ultralow temperatures. The apparatus also includes a programmable-temperature chamber to control the rates of chemical reaction. For chemical fixation, cells are treated with 0-4% formaldehyde, washed, and then treated with protein-reactive reagents. Cells are imaged by wide-field fluorescence microscopy, using fluorescent markers for various cell structures. Standard 1D and 2D proteomics methods were used after labeling, with an Eclipse Mass Spectrometer fronted by a Vanquish Neo nanoLC C18 separation system. Data were analyzed with MSFragger for proteome coverage and monolink detection, and by CRIMP 2.0 for crosslink detection.
Preliminary data: The cryofixation process was optimized to ensure that freeze-substitution would not disturb cellular ultrastructure. For example, we show the expected phalloidin staining patterns of the actin cytoskeleton, which demands preservation of actin’s 3D structure, and the expected DAPI staining patterns of an undistorted nucleus. We then surveyed an extensive list of amine-selective chemistries (in “monovalent” form) to determine the foundation for an effective crosslinker for low-temperature labeling. Ortho-phthaldehydes demonstrate the best balance of selectivity, water resistance (for ease of handling) and reactivity at low temperatures. For example, we show that greater than 15% of all cellular lysines could be labeled at -40°C with simple “monovalent” ortho-phthaldehydes. We then synthesized a series of di-ortho-phthalaldehydes (DOPAs), designed for high solubility in cold acetone and effective interaction sampling, and tested them on frozen cells. Data analysis of “DOPA4” through an initial shotgun LC-MS/MS experiment from E. coli cells demonstrated extensive crosslinking. Parallel control XL-MS experiments using the DSS crosslinker in a conventional in situ manner demonstrated fewer CSMs. Digest fractionation and experiments with enrichable DOPAs are ongoing. An alternative fixation procedure involves formaldehyde pre-treatment, long demonstrated to preserve cell structure. Surprisingly, we show that formaldehyde-fixed cells support extensive monovalent chemical labeling of lysines. Labeling yields actually increase, plateauing between 1 and 4% formaldehyde pretreatment (a conventional fixation recipe). Amine-based crosslinkers work equally well when applied to formalin-fixed cells, generating high quality CSMs. In the talk, we will demonstrate how fixation allows for alternative crosslinker reaction schemes, including membrane permeabilization to introduce normally non-permeable reagents, and multiple applications of reagent to boost signal, to achieve full control over the crosslinking reaction.
Novel aspect: Prefixing cells preserves cell structure, restoring organizational fidelity to XL-MS experiments and expanding the potential of the technique.
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