ABSTRACT Background Proteolytic processing is a fundamental post‐translational modification. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) workflows are powerful for degradomic analyses but inherently sacrifice spatial information, a critical aspect for investigating biological systems such as aberrant extracellular matrix remodeling and alterations of the tumor microenvironment. Matrix‐assisted laser desorption/ionization (MALDI) offers potential for fast spatial profiling, but MALDI imaging of tryptic peptides is still challenged by spectral crowding and restricted abilities for MALDI MS/MS identification. Methods To address these limitations, we developed pyrene‐conjugated 2‐pyridinecarboxaldehyde (pyr‐2PCA) tags for selective N‐terminal labeling and enhanced detection sensitivity. The 2PCA reagent exclusively modifies N‐terminal α‐amines, not lysine ε‐amines, as could be confirmed in MALDI‐MS, with concentration‐dependent side reactions minimized by dilution. A distinct reporter ion produced by 2PCA‐labeled peptides in prm‐PASEF (MALDI MS/MS) serves as a unique marker for successful labeling. Results The covalent conjugation of 2PCA with a pyrene structure results in the pyr‐2PCA tag that enables matrix‐free, label‐assisted laser desorption ionization mass spectrometry (LALDI‐MS) measurements of peptides. We demonstrate that labeling with a pyrene‐coupled 2PCA tag (pyr‐2PCA) prior to tryptic digestion results in the selective detection of N‐terminal peptides in LALDI, with no significant off‐target labeling. Conclusions This study presents the first presentation and characterization of this novel pyr‐2PCA tag, thereby laying the groundwork and demonstrating its future potential for MALDI/LALDI‐based in situ spatial N‐terminomics to study proteolytic processes.
Li et al. (Tue,) studied this question.