Figure 2.

Simplified schemes for the major methods used in micro-level anesthetic photoaffinity labeling; a) Edman degradation b) Tandem mass spectrometry (MS/MS). a) Edman degradation; i_a) enriched target protein is photoaffinity labeled with radiolabeled (*) anesthetic photoaffinity ligand (PAL) and protein(s) are separated and digested into peptides; ii_a) peptides are isolated and rigorously purified; iii_a) the first amino acid from the N-termini is cleaved from purified peptide via Edman reaction; iv_a) the cleaved amino acid is isolated, quantified and separated into two pools for (top) radioisotope detection by scintillation counting and (bottom) amino acid identification by chromatography; v_a) the cycle is repeated, gradually sequencing the entire purified peptide; vi_a) the amino acid sequence is plotted against radioactivity (cpm, counts per minute, or dpm, disintegrations per minute) resulting in radioisotope release profile. b) Tandem mass spectrometry (MS/MS); i_b) enriched target protein is photoaffinity labeled with anesthetic photoaffinity ligand (PAL) and protein(s) are separated and digested into peptides that are further separated by on- and/or offline chromatography methods; ii_b) peptides undergo ionization generally by electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI); iii_b) precursor ions are separated by mass/charge (m/z) within MS1 (aka. MS1 precursor ion); iv_b) MS1 precursor ion undergoes mass fragmentation often by collision-induced dissociation (CID) resulting in largely b or y fragment ions; v_b) mass spectrometry software are used for data acquisition, database search analysis and representation.