Targeted Single-Cell Microchemical Analysis: MS-Based Peptidomics of Individual Paraformaldehyde-Fixed and Immunolabeled Neurons

SummaryPinpointing a specific cell from within a relatively uniform cell population to determine its chemical content presents a challenging bioanalytical task. Immunocytochemistry is the classical method used to localize specific molecules and, hence, selected cells. Mass spectrometry also probes endogenous molecules such as neuropeptides within a cell. Here, these two approaches are hyphenated to allow microchemical analysis of immunocytochemical-selected peptidergic neurons. This two-step strategy utilizes antibody-based localization of cells containing selected biomarkers to isolate the cell(s) of interest, followed by peptidomic analysis via mass spectrometry. Applicable to a broad range of analyte and cell types, the strategy was used to successfully profile neuropeptides from individual immunostained insect neurons stored for up to 2 weeks as well as from tissues preserved for 42 weeks.

Graphical AbstractFigure optionsDownload high-quality image (153 K)Download as PowerPoint slideHighlights
► Investigation of archived biological tissue at the single-cell level
► Screening of neuropeptides from immunolabeled tissue preserved up to 42 weeks
► Validation of antibody specificity using mass spectrometric approaches
► Standardized protocols for comprehensive characterization of selected cells