Discovery and Identification of Scarce Terminal Alkyne-Containing Natural Products by Chemical Isotope Labeling-Assisted MALDI-TOF Mass Spectrometry.

Terminal alkyne-containing natural products (TANPs) are pharmacologically valuable but remain underexplored due to their low abundance, instability, and inefficient isolation methodologies. Herein, we employed a structure-guided screening and isolation strategy integrating chemical isotope labeling (CIL) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for TANPs discovery. Parallel plant extracts were labeled via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction using isotope labeling reagents ABI/d5-ABI, followed by MALDI-TOF MS analysis. This process generated stable labeled products with enhanced detection sensitivity and two distinctive features as screening criteria: (i) isotope peak pairs (Δm/z = 5.0314 Da) and (ii) N2 neutral loss (28.0056 Da), enabling rapid TANPs identification. Comparative chromatographic analysis of labeled/unlabeled samples allowed for oriented TANPs isolation. Guided by this method, five TANPs were successfully recognized, isolated, and identified from Spilanthes callimorpha for the first time, including a novel glycoside-containing compound. This CIL-MALDI-TOF MS method provides an efficient alternative for the discovery of TANPs in complex extracts.