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.
Highlights
Organelles are one of the smallest structural units that influence the functional, morphological and biochemical characteristics of different cell types
Our method revealed post-translational proteolytic processing of AGPB1 (XP_012945142.1), AGPA1 (XP_012945143.1) and AGPA2 (XP_012945134.1) prohormones, with peptide structures validated by liquid chromatography–tandem mass spectrometry (LC–Mass spectrometry (MS)/MS)
By measuring the pixel properties produced by dense-core vesicles (DCVs) under brightfield conditions, we compiled sequences of image algorithms (Supplementary Fig. 4) that selectively identify the pixels produced by DCVs versus the background (Fig. 1b–d), allowing identification of the spatial locations of individual DCVs without the need for chemical labeling
Summary
Organelles are one of the smallest structural units that influence the functional, morphological and biochemical characteristics of different cell types. Mass spectrometry (MS) imaging has begun being used for both cellular and some subcellular analyses in discovery-based studies but is limited in throughput and spatial resolution for organelle measurements Both cells and organelles of interest can be isolated and placed on a glass slide for subsequent MS measurement. We adapted the single-cell approach to single organelles, which required three enhancements: (1) improved object targeting approaches, (2) optimized analyte detection using high-resolution MS and (3) unsupervised data analysis workflows to characterize organelle heterogeneity These advances allowed us to use MALDI Fourier-transform ion cyclotron resonance MS for the high-throughput simultaneous detection of both peptide and lipid species in 0.5- to 2-μm-diameter dense-core vesicles (DCVs) and electron lucent vesicles (LVs) isolated from the exocrine atrial gland (AG) (Fig. 1a–f) and red hemiduct of Aplysia californica, respectively. Our workflow is extendable to multiple imaging modalities such as scanning electron microscopy (Supplementary Fig. 3) and instrumentation that uses a piezo linear stage and camera, providing an avenue for the analysis of targets that are smaller than the wavelength of light
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