By incorporating a high proton affinity moiety to the charge localized free radical-initiated peptide sequencing (CL-FRIPS) reagent, FRIPS-MS technique has extended the applicability to hydrophobic peptides and peptides without basic amino acid residues (lysine, arginine, and histidine). Herein, the CL-FRIPS reagent has three moieties, 1) pyridine acting as the basic site to locate the proton, 2) 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO, a stable free radical) acting as the free radical precursor to generate the nascent free radical in the gas phase, and 3) Nhydroxysuccinimide (NHS) activated carboxylic acid acting as the coupling site to derivatize the N-terminus of peptides. The CL-FRIPS reagent allows for the characterization of peptides by generating sequencing ions, enzymatic-cleavage-like radical-induced side chain losses, and the loss of TEMPO simultaneously via one-step collisional activation. Further collisional activation of enzymatic-cleavage-like radical-induced side chain loss ions provides more information for the structure determination of peptides. The application of CL-FRIPS reagent to characterize peptides is proved by employing bovine insulin as the model peptide. Both scaffold structure of bovine insulin and sequencing information of each chain are achieved. GRAPHICAL ABSTRACTS INTRODUCTION Gas-phase free radical/electron techniques combined with mass spectrometry has recently gained significant interest in the field of characterization of biological macromolecules,

Inspired by the electron-activated dissociation technique, the most potent tool for glycan characterization, we recently developed free radical reagents for glycan structural elucidation. However, the underlying mechanisms of free radicalinduced glycan dissociation remain unclear and, therefore, hinder the rational optimization of the free radical reagents and the interpretation of tandem mass spectra, especially the accurate assignment of the relatively low-abundant but information-rich ions. In this work, we selectively incorporate the 13 C and/or 18 O isotopes into cellobiose to study the mechanisms for free radicalinduced dissociation of glycans. The eight isotope-labeled cellobioses include 1-13 C, 3-13 C, 1′-13 C, 2′-13 C, 3′-13 C, 4′-13 C, 5′-13 C, and 1′-13 C−4-18 O-cellobioses. Upon one-step collisional activation, cross-ring (X ions), glycosidic bond (Y-, Z-, and B-related ions), and combinational (Y 1 + 0,4 X 0 ion) cleavages are generated. These fragment ions can be unambiguously assigned and confirmed by the mass difference of isotope labeling. Importantly, the relatively low-abundant but information-rich ions, such as