Collection Citation: Sayan Dutta, Marion Pang, Gerard Coughlin 2026. Molecularly-guided spatial proteomics captures single-cell identity of the healthy and diseased nervous system. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn1j4yv5d/v1
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: February 13, 2026
Last Modified: March 31, 2026
Collection Integer ID: 243303
Keywords: guided spatial proteomic, cell spatial proteomic, neuronal proteomic result, spatial proteomic, specific neuronal proteome, proteomic differences between dopaminergic neuron subpopulation, heterogeneous tissues such as the brain, proteomic difference, proteome coverage, bearing single dopaminergic neuron, single dopaminergic neuron, complementary transcriptomic resource, peripheral somatic tissue, diseased mammalian brain, laser capture microdissection, unbiased mass spectrometry, protein signal
Abstract
Single-cell spatial proteomics (scSP) holds substantial potential for profiling healthy and
diseased tissues. The emerging method of molecularly-guided unbiased scSP has mostly
been applied to peripheral somatic tissues. Here, we optimize and apply untargeted
scSP to the healthy and diseased mammalian brain, using molecularly-guided
laser capture microdissection and unbiased mass spectrometry. We systematically
evaluate the effects of tissue fixation, marker staining, and sample input size
on proteome coverage and quantitative accuracy. We benchmark this workflow by
profiling region-specific neuronal proteomes and mapping the response of
non-neuronal cells to acute brain injury. Across these applications, we
integrate complementary transcriptomic resources to evaluate cross-modality
trends and refine neuronal proteomic results by filtering out protein signals
likely arising from non-neuronal cells, an essential consideration in
heterogeneous tissues such as the brain. Finally, we leverage this approach to
resolve proteomic differences between dopaminergic neuron subpopulations with
differential vulnerability to Parkinson’s disease and to uncover disease-specific
disruptions in α-synuclein-aggregate-bearing single dopaminergic neurons.
Together, these data demonstrate the utility of scSP in neuroscience research
for understanding fundamental biology and the molecular drivers of neurological
conditions.
Guidelines
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