Optically Guided Single Cell Mass Spectrometry of Rat Dorsal Root Ganglia to Profile Lipids, Peptides and Proteins

Abstract

The mammalian dorsal root ganglia (DRG) are located on the dorsal roots of the spinal nerves and contain cell bodies of primary sensory neurons. DRG cells have been classified into subpopulations based on their size, morphology, intracellular markers, response to stimuli, and neuropeptides. To understand the connections between DRG chemical heterogeneity and cellular function, we performed optically guided, high-throughput single cell profiling using sequential matrix-assisted laser desorption/ionization mass spectrometry (MS) to detect lipids, peptides, and several proteins in individual DRG cells. Statistical analysis of the resulting mass spectra allows stratification of the DRG population according to cellular morphology and, presumably, major cell types. A subpopulation of small cells contained myelin proteins, which are abundant in Schwann cells, and mass spectra of several larger cells contained peaks matching neurofilament, vimentin, myelin basic protein S, and thymosin beta proteins. Of the over 1000 cells analyzed, approximately 78 % produced putative peptide-rich spectra, allowing the population to be classified into three distinct cell types. Two signals with m/z 4404 and 5487 were exclusively observed in a cell type, but could not be matched to results of our previous liquid chromatography-MS analyses.

Lipid Heterogeneity between Astrocytes and Neurons Revealed by Single‐Cell MALDI‐MS Combined with Immunocytochemical Classification

“…[6] While matrix-assisted laser desorption/ionization (MALDI) MS produces rich spectral profiles for label-free classifications, [7] the results can be difficult to correlate to canonical cell types. [8] Immunocytochemistry (ICC) is effective for classifying cells as it can target ap lethora of different molecules that distinguish cell types. [9] ICC has been combined with tissue imaging and af ew examples have coupled the method to single-cell MS. [10] However,f luorescence overlap of labels typically limits plexity to fewer than ten compounds, [11] and most ICC protocols require sample fixation and lipid removal, crosslinking and/or removing many compounds that are easily ionized by MALDI-MS. [12] Direct hyphenation of ICC and MS to analyze the same cell was reported in 2012, [10b] but ICC was performed first, limiting the analysis to high-abundance peptides within as mall number of cells.H erein, we describe aworkflow enabling high-throughput MALDI-MS analysis of thousands of cells,followed by cell classification through ICC.…”

Approaching sites of action of drugs in clinical pharmacology: New analytical options and their challenges

“…127 Laser desorption ionization (LDI) 128 and MALDI have also been used for single cell analysis. 124,[129][130][131] Conductive slides coated with hydrophobic laser-drilled cavities were used to organize cell smears in arrays (called microarrays for MS or MAMS), which were subsequently analysed by MALDI profiling of single cells. 129 Combined with automated smearing methods to deposit drug solution on surfaces, it has been demonstrated that MAMS would hold good promise for drug quantification.…”

Exploring the Fundamental Structures of Life: Non‐Targeted, Chemical Analysis of Single Cells and Subcellular Structures

“…To expand optically guided, single-cell profiling beyond MALDI-TOFi nstrumentation, Comi et al [115] further developed the open source software,m icroMS,f or analyzing microscopy images and correlating cell locations to instrument positions.T he software provides automatic cell finding and filtering by attributes such as size,fluorescence intensity, and distance between cells,and is simple to tailor to avariety of instruments,e xpanding the availability of single-cell profiling.T he ease of switching between output coordinate systems also facilitates analysis of the same cell on multiple instruments;a ne xample being MALDI-TOFM Sa nalysis with follow-up CE-MS analysis ( Figure 6D). [115] Do et al [116] recently used microMS to set up sequential MALDI-MS analyses of rat DRG cells.Lipids,peptides,and small proteins were detected in the same cells,m any of which were not previously detected in tissue homogenates or releasates.T he DRG populations were stratified with multivariate statistical analysis using peptides and proteins as potential markers.A similar method utilizing flexImaging software from Bruker Corp.w as recently published by the Caprioli lab [117] to determine heterogeneity within cultured macrophage cells using aB ruker solariX XR FT-ICR mass spectrometer.T he authors imaged single cells by rastering across the slide and comparing the ion images with optical images acquired beforehand. They observed changes in lipid expression of macrophages upon chemical stimulation.…”