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,

The inherent structural complexity and diversity of glycans pose a major analytical challenge to their structural analysis. Radical chemistry has gained considerable momentum in the field of mass spectrometric biomolecule analysis, including proteomics, glycomics, and lipidomics. Herein, seven isomeric disaccharides and two isomeric tetrasaccharides with subtle structural differences are distinguished rapidly and accurately via one-step radical-induced dissociation. The free-radical-activated glycan-sequencing reagent (FRAGS) selectively conjugates to the unique reducing terminus of glycans in which a localized nascent free radical is generated upon collisional activation and simultaneously induces glycan fragmentation. Higher-energy collisional dissociation (HCD) and collision-induced dissociation (CID) are employed to provide complementary structural information for the identification and discrimination of glycan isomers by providing different fragmentation pathways to generate informative, structurally significant product ions. Furthermore, multiple-stage tandem mass spectrometry (MS 3 CID) provides supplementary and valuable structural information through the generation of characteristic parent-structure-dependent fragment ions.

By combing the merits of solid supports and free radical activated glycan sequencing (FRAGS) reagents, we develop a multi-functional solid-supported free radical probe (SS-FRAGS), which enables glycan enrichment and characterization. SS-FRAGS comprises a solid support, free radical precursor, disulfide bond, pyridyl, and hydrazine moieties. Thioactivated resin and magnetic nanoparticles (MNPs) are chosen as the solid support to selectively capture free glycans via the hydrazine moiety, allowing for their enrichment and isolation. The disulfide bond acts as a temporary covalent linkage between the solid support and the captured glycan, allowing the release of glycans via the cleavage of the disulfide bond by dithiothreitol. The basic pyridyl functional group provides a site for the formation of a fixed charge, enabling detection by mass spectrometry and avoiding glycan rearrangement during collisional activation. The free radical precursor generates a nascent free radical upon collisional activation and thus simultaneously induces systematic and predictable fragmentation for glycan structure elucidation. A radical-driven glycan deconstruction diagram (R-DECON) is developed to visually summarize the MS 2 results and thus allow for the assembly of the glycan skeleton, making the differentiation of isobaric glycan isomers unambiguous. For application to a real-world sample, we demonstrate the efficacy of the SS-FRAGS by analyzing glycan structures enzymatically cleaved from RNase-B.

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