A newly developed mass spectrometry imaging protocol can map intact protein forms in biological tissue at a spatial resolution of approximately 10 micrometers, according to a recent study.
Proteoforms are distinct versions of a protein created through processes such as genetic variation, alternative splicing, and post-translational modification. Their location within tissue can provide information that is lost when samples are homogenized for conventional proteomic analysis.
Manxi Yang and colleagues of the James Tarpo Jr. and Margaret Tarpo Department of Chemistry at Purdue University described a detailed workflow using nanospray desorption electrospray ionization, or nano-DESI, mass spectrometry imaging.
Nano-DESI uses two small glass capillaries to form a continuously flowing liquid bridge on the tissue surface. One capillary delivers an extraction solvent, while the second transfers the extracted molecules to the mass spectrometer. Moving the tissue beneath the probe line by line creates a molecular map of the sampled region.
Before imaging, the protocol uses a series of ethanol and chloroform washes to remove lipids from fresh-frozen tissue sections. Lipids ionize efficiently and can suppress protein signals. The ethanol washes also precipitate proteins within the tissue, helping to limit their movement during delipidation.
A shear-force probe maintains a constant distance between the nano-DESI probe and the tissue. This helps stabilize the liquid bridge and prevents the sampling capillaries from touching or damaging the surface. The authors used a sampling tip of approximately 10 micrometers, a stage speed of around 10 micrometers per second and line spacing of approximately 7 micrometers.
The extraction solvent contained acetonitrile, water, and formic acid. Proteins extracted directly from the tissue produced multiply charged ions, which are suitable for top-down tandem mass spectrometry. Proteoforms were identified by comparing their intact masses and fragmentation patterns with database records.
Representative images from mouse brain tissue showed the spatial distributions of several proteoforms, including modified forms of myelin basic protein, acyl-CoA-binding protein, cytochrome c oxidase subunit 6B1, and cytoplasmic dynein light chain 2. Targeted tandem mass spectrometry can be performed on an adjacent tissue section after imaging identifies regions of interest.
The method provides spatial information on intact proteins and their modifications without requiring labels. It could support studies examining how closely related proteoforms are distributed across different tissue structures.
Current nano-DESI experiments typically detect tens of proteoforms, not the full tissue proteome. Most reported targets are abundant proteins smaller than 20 kilodaltons, although larger proteins have been measured in other studies. The authors’ previous work mapped approximately 40 proteoforms representing 19 proteins in rat brain tissue.
Higher spatial resolution also reduces the amount of material collected from each location, weakening protein signals. Tissue thickness can partially offset this loss, but thicker sections may make the surface less uniform and increase the risk of blocking the small capillaries. The authors identified sensitivity, ionization efficiency, spectral processing, and fragmentation as areas for further improvement.
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