Single Cell Peptidomics: Approach for Peptide Identification by N-Terminal Peptide Derivatization

In recent years, single cell microanalysis techniques have moved into the center stage to study fundamental intracellular interactions and cell-cell communication events, and have led to a better understanding of physiological processes and behavioral patterns. The availability of more sensitive, robust, and precise mass spectrometers improved the detection and characterization of putative neuroactive substances from individual cells. For sequence characterization, particularly when working with samples as small as a single cell, the most crucial step to obtain usable data is sample preparation. For some studies, genetic or molecular data are not available to confirm an amino acid sequence of a putative neuropeptide, and it is necessary to sequence the peptide from the mass spectrometry analysis alone (i.e., de novo sequencing). In this chapter, a protocol is described for de novo sequencing of neuropeptides from individual single cells by N-terminal derivatization using 4-sulfophenyl isothiocyanate and subsequent mass spectrometric analysis.