Subcellular Peptide Localization in Single Identified Neurons by Capillary Microsampling Mass Spectrometry

Abstract

Single cell mass spectrometry (MS) is uniquely positioned for the sequencing and identification of peptides in rare cells. Small peptides can take on different roles in subcellular compartments. Whereas some peptides serve as neurotransmitters in the cytoplasm, they can also function as transcription factors in the nucleus. Thus, there is a need to analyze the subcellular peptide compositions in identified single cells. Here, we apply capillary microsampling MS with ion mobility separation for the sequencing of peptides in single neurons of the mollusk Lymnaea stagnalis, and the analysis of peptide distributions between the cytoplasm and nucleus of identified single neurons that are known to express cardioactive Phe-Met-Arg-Phe amide-like (FMRFamide-like) neuropeptides. Nuclei and cytoplasm of Type 1 and Type 2 F group (Fgp) neurons were analyzed for neuropeptides cleaved from the protein precursors encoded by alternative splicing products of the FMRFamide gene. Relative abundances of nine neuropeptides were determined in the cytoplasm. The nuclei contained six of these peptides at different abundances. Enabled by its relative enrichment in Fgp neurons, a new 28-residue neuropeptide was sequenced by tandem MS.

Optical Microscopy-Guided Laser Ablation Electrospray Ionization Ion Mobility Mass Spectrometry: Ambient Single Cell Metabolomics with Increased Confidence in Molecular Identification

“…A comparison of the number of spectral features ( Figure 5 c), and the number of an annotated species between the bulk and single cell spectra ( Figure 5 d) illustrates that single cell analysis produces greater molecular coverage than bulk analysis; with saccharides being the main molecular class detected in both sampling methods. This difference in metabolite profiles can be attributed to a combination of several factors, which include dilution of specific metabolites by non-metabolite containing cells in the bulk extract [ 54 ], insolubility of metabolites detected in situ in the aqueous phase of the MPLEx extract [ 55 ], cellular heterogeneity and metabolite heterogeneity across the cell population, as well as charge competition in ESI [ 56 ].…”

Single organelle function and organization as estimated from Arabidopsis mitochondrial proteomics

“…Heterogeneity in mitochondrial populations likely carries biological significance, especially for those proteins present at very low or even substoichiometric copy numbers. Micromanipulation‐ or MALDI imaging‐based approaches to resolve individual mitochondria may provide an avenue for proteins with larger copy numbers and favourable properties for mass spectrometry analysis (Yajima et al ., ; Zhang et al ., ). Innovative single cell proteomic approaches and single molecule approaches, such as nanopore sequencing of proteins (Budnik et al ., ; Restrepo‐Pérez et al ., ) may open the door to the first single organelle proteomes with quantitative protein coverage.…”

Cell-Type-Specific Proteomics: A Neuroscience Perspective

“…In the same year, they also reported identification of a total of 1709 protein groups from 20 ng of Xenopus protein digest from three cell types of the 16-cell embryo [133]. In addition to electrophoresis, capillaries have recently been used for microsampling of biomolecules from single neurons [140]. This study integrated this technique with downstream ESI-IMS-MS, which had only previously been performed in human carcinoma cells [141] and Arabidopsis thaliana epidermal cells [142].…”