A Glycosidic‐Bond‐Based Mass‐Spectrometry‐Cleavable Cross‐linker Enables In Vivo Cross‐linking for Protein Complex Analysis

Abstract Chemical cross‐linking mass spectrometry (CXMS) has emerged as a powerful technology to analyze protein complexes. However, the progress of in vivo CXMS studies has been limited by cross‐linking biocompatibility and data analysis. Herein, a glycosidic bond‐based MS‐cleavable cross‐linker of trehalose disuccinimidyl ester (TDS) was designed and synthesized, which was fragmented in MS under CID/HCD to simplify the cross‐linked peptides into conventional single peptides via selective cleavage between glycosidic and peptide bonds under individual MS collision energy. Consequently, the cross‐linking identification accuracy and throughput were significantly enhanced, and the popular MS mode of stepped HCD was allowed. In addition, TDS showed proper cell‐penetrating properties while being highly water‐soluble, making it non‐DMSO dependent during solubilization. Collectively, TDS provides a promising toolkit for CXMS characterization of living systems with high biocompatibility and accuracy.