Image-guided MALDI mass spectrometry for high-throughput single-organelle characterization

Abstract

Peptidergic dense-core vesicles are involved in packaging and releasing neuropeptides and peptide hormones—critical processes underlying brain, endocrine and exocrine function. Yet, the heterogeneity within these organelles, even for morphologically defined vesicle types, is not well characterized because of their small volumes. We present image-guided, high-throughput mass spectrometry-based protocols to chemically profile large populations of both dense-core vesicles and lucent vesicles for their lipid and peptide contents, allowing observation of the chemical heterogeneity within and between these two vesicle populations. The proteolytic processing products of four prohormones are observed within the dense-core vesicles, and the mass spectral features corresponding to the specific peptide products suggest three distinct dense-core vesicle populations. Notable differences in the lipid mass range are observed between the dense-core and lucent vesicles. These single-organelle mass spectrometry approaches are adaptable to characterize a range of subcellular structures.

Profiling 26,000 Aplysia californica neurons by single cell mass spectrometry reveals neuronal populations with distinct neuropeptide profiles

“…First, we used the most common MALDI mass analyzer, the TOF, as it demonstrates that the approach can be used with equipment found in facilities at most universities. However, this limits the mass resolution and MS/MS abilities (for high-confidence peptide assignments); if higher-quality spectra are required or peptide sequencing is needed, Orbitrap ( 10 , 12 ), ion cyclotron resonance ( 40 , 41 , 42 ), or timsTOF ( 97 , 98 ) instruments are also amenable to the sampling and informatics workflow. Second, while this protocol is effective at detecting the peptides within cells, a mass spectrometer will measure whatever is at the specific location on the slide.…”

“…We have created a MALDI MS workflow that, when applied to low density, dissociated single cells characterized by microscopy, acquires single cell mass spectra within several seconds ( 36 ), allowing thousands of cells to be profiled in a single experiment ( 37 , 38 , 39 ). Though these prior single cell MALDI MS studies focused on the characterization of lipids in tens of thousands of cells ( 38 , 40 ) and produced robust data that can be used to classify cells ( 41 ), the approach has also been used to characterize single organelles ( 42 ) and individual cells from endocrine structures, including islets and the pituitary from rats ( 19 , 43 ).…”

Emerging Computational Methods in Mass Spectrometry Imaging

“…This strategy has been recently used for the high‐throughput single‐organelle characterization with subcellular precision. [ 254 ]…”

“…This strategy has been recently used for the high-throughput single-organelle characterization with subcellular precision. [254] Compressed sensing is another "smart" sampling strategy that does not require external input. Compressed sensing is an approach, which relies on sparse sampling from selected locations on a sample followed by image reconstruction using a uniform virtual grid.…”

Space and Time Coherent Mapping for Subcellular Resolution of Imaging Mass Spectrometry

“…The spatial resolution of the image depends on the probe size and sampling distance, and it typically ranges from several to over tens of micrometers, which is often too coarse for cellular levels of imaging. To circumvent the limitation in spatial resolution, subcellular organelles such as lysosomes and dense core vesicles were isolated and laid out densely in vitro for spot-targeted IMS [ 1 , 2 ]. Although the strategy helped image subcellular organelles, it cannot be used for high-resolution imaging in samples with complex multi-cellular organizations typically found in the human brain, even if software tools are in place for the automated processing of huge datasets.…”