Single cell analysis strives to probe molecular heterogeneity in morphologically similar cell populations through quantitative or qualitative measurements of genetic, proteomic, or metabolic ...products. Here, we applied mass analysis of single neurons to investigate cell–cell signaling peptides. The multiplicity of endogenous cell–cell signaling peptides is a common source of chemical diversity among cell populations. Certain peptides can undergo post-translational isomerization of select residues, which has important physiological consequences. The limited number of single cell analysis techniques that are sensitive to peptide stereochemistry make it challenging to study isomerization at the individual cell level. We performed capillary electrophoresis (CE) with mass spectrometry (MS) detection to characterize the peptide content of single cells. Using complementary trapped ion mobility spectrometry (TIMS) separations, we measured the stereochemical configurations of three neuropeptide gene products derived from the pleurin precursor in individual neurons (N = 3) isolated from the central nervous system of Aplysia californica. An analysis of the resultant mobility profiles indicated >98% of the detectable pleurin-derived peptides exist as the nonisomerized, all-l forms in individual neuron cell bodies. However, we observed 44% of the Plrn2 peptide from the pleurin precursor was present as the isomerized, d-residue-containing form in the nerve tissue. These findings demonstrate an unusual distribution of isomerized peptides in A. californica and establish CE–TIMS MS as a powerful analytical tool for investigating peptide stereochemistry at the single cell level.
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.
Acetylcholine, the first neurotransmitter identified more than a century ago, plays critical roles in human activities and health; however, its synaptic concentration dynamics have remained unknown. ...Here, we demonstrate the in situ simultaneous measurements of synaptic cholinergic transmitter concentration and release dynamics. We used nanoscale electroanalytical methods: nanoITIES electrode of 15 nm in radius and nanoresolved scanning electrochemical microscopy (SECM). Time-resolved in situ measurements unveiled information on synaptic acetylcholine concentration and release dynamics of living Aplysia neurons. The measuring technique enabled the quantitative sensing of acetylcholine with negligible interference of other ionic and redox-active species. We measured cholinergic transmitter concentrations very close to the synapse, with values as high as 2.4 mM. We observed diverse synaptic transmitter concentration dynamics consisting of singlet, doublet and multiplet events with a signal-to-noise ratio of 6 to 130. The unprecedented details about synaptic neurotransmission unveiled are instrumental for understanding brain communication and diseases in a way distinctive from extra-synaptic studies.
Transcriptomics characterizes cells based on their potential molecular repertoire whereas direct mass spectrometry (MS) provides information on the actual compounds present within cells. Single‐cell ...matrix‐assisted laser desorption/ionization (MALDI) MS can measure the chemical contents of individual cells but spectra are difficult to correlate to conventional cell types, limiting the metabolic information obtained. We present a protocol that combines MALDI‐MS with immunocytochemistry to assay over a thousand individual rat brain cells. The approach entwines the wealth of knowledge obtained by immunocytochemical profiling with mass spectral information on the predominant lipids within each cell. While many lipid species showed a high degree of similarity between neurons and astrocytes, seventeen significantly differed between the two cell types, including four phosphatidylethanolamines elevated in astrocytes and nine phosphatidylcholines in neurons.
Single‐cell lipidomics: The combination of single‐cell MALDI‐MS and immunocytochemistry allows metabolic information to be directly coupled to canonical cell type. Analysis of over a thousand single cells shows that many lipid species have a high similarity between neurons and astrocytes, although seventeen lipid species differed significantly between the two cell types, including four phosphatidylethanolamines elevated in astrocytes and nine phosphatidylcholines in neurons.
d-Alanine (d-Ala) is an unusual endogenous amino acid present in invertebrates and vertebrates. Compared to its l-isomer, the characterization of d-Ala is challenging because of the need for chiral ...resolution and the low amounts of the d-enantiomer present. With recent improvements in measurement capabilities, research on d-Ala, along with other d-amino acids, has been growing, especially as the functional significance of d-Ala in the mammalian nervous and endocrine systems is becoming known. Here we provide an overview of the distribution, origin, function, and disease implications of d-Ala.
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•d-alanine is widely distributed in invertebrates and vertebrates.•d-alanine originates from diet, microbiota, and potentially endogenous synthesis.•Acts as a putative signaling molecule in the nervous and endocrine systems.•Physiological relevance in the gut-microbiota-brain axis and in the immune response.•d-alanine levels track various disease states suggesting a potential therapeutic role.
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