Native Mass Spectrometry Imaging and In Situ Top-Down Identification of Intact Proteins Directly from Tissue.

Oliver J Hale,Helen J Cooper

doi:10.1021/jasms.0c00226

Oliver J Hale, Helen J Cooper

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https://doi.org/10.1021/jasms.0c00226

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Mass spectrometry imaging (MSI) provides information on the spatial distribution of molecules within a biological substrate without the requirement for labeling. Its broad specificity, i.e., the capability to spatially profile any analyte ion detected, constitutes a major advantage over other imaging techniques. A separate branch of mass spectrometry, native mass spectrometry, provides information relating to protein structure through retention of solution-phase interactions in the gas phase. Integration of MSI and native mass spectrometry ("native MSI") affords opportunities for simultaneous acquisition of spatial and structural information on proteins directly from their physiological environment. Here, we demonstrate significant improvements in native MSI and associated protein identification of intact proteins and protein assemblies in thin sections of rat kidney by use of liquid extraction surface analysis on a state-of-the-art Orbitrap mass spectrometer optimized for intact protein analysis. Proteins of up to 47 kDa, including a trimeric protein complex, were imaged and identified.

Highlights

Mass spectrometry imaging (MSI) enables molecules to be spatially mapped throughout a biological substrate, such as a thin tissue section.[1]
Our long-term goal is to combine the benefits of MSI and native MS, i.e., to obtain information on both spatial distribution and tertiary or quaternary structure, through native mass spectrometry imaging
Major urinary protein (MUP), K-FABP, and RidA were detected in the cortex, whereas H-FABP was detected in the medulla

Summary

■ INTRODUCTION

Mass spectrometry imaging (MSI) enables molecules to be spatially mapped throughout a biological substrate, such as a thin tissue section.[1]. Ion detection was performed in the Orbitrap mass analyzer, operating at a resolution of 15 000 (at m/z 200) for imaging experiments and 60 000−500 000 as required for additional experiments (high-resolution full scan spectra, MSn). MSn. Protein ions were selected for MSn on the basis of their abundance following manual inspection of the mass spectra in the imaging data set. MSn of abundant ions (RidA10+, holo-alphaglobin5+) was performed directly from a single sampling location with spectra obtained in less than 5 min. Summed mass spectra for each pixel were generated in FreeStyle (version 1.4, Thermo Scientific) and exported in the Thermo raw format These files were converted to mzML by msconvert (Version 3.0, ProteoWizard Software Foundation).[13] The image file was produced with imzML converter (version 1.3).[14] All mzML files were imported and processed with the “pixel per file” option. Tentative identifications were provided by ProSight, with further assignment of MSn signals performed manually using MS-Product (ProteinProspector, v 5.24.0, http://prospector. ucsf.edu/prospector/mshome.htm, UCSF) to predict fragment m/z

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■ CONCLUSIONS

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