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.

Native top-down proteomics allows for both proteoform identification and high-order structure characterization for cellular protein complexes. Unfortunately, tandem MS-based fragmentation efficiencies for such targets are low due to an increase in analyte ion mass and the low ion charge states that characterize native MS data. Multiple fragmentation methods can be integrated in order to increase protein complex sequence coverage, but this typically requires use of specialized hardware and software. Free-radical-initiated peptide sequencing (FRIPS) enables access to charge-remote and electron-based fragmentation channels within the context of conventional CID experiments. Here, we optimize FRIPS labeling for native top-down sequencing experiments. Our labeling approach is able to access intact complexes with TEMPO-based FRIPS reagents without significant protein denaturation or assembly disruption. By combining CID and FRIPS datasets, we observed sequence coverage improvements as large as 50% for protein complexes ranging from 36 to 106 kDa. Fragment ion production in these experiments was increased by as much as 102%. In general, our results indicate that TEMPObased FRIPS reagents have the potential to dramatically increase sequence coverage obtained in native top-down experiments.

Despite recent advances in glycomics, glycan characterization still remains an analytical challenge. Accordingly, numerous glycan‐tagging reagents with different chemistries were developed, including those involving acid‐base chemistry and/or free radical chemistry. Acid‐base chemistry excels at dissociating glycans into their constituent components in a systematic and predictable manner to generate cleavages at glycosidic bonds. Glycans are also highly susceptible to depolymerization by free radical processes, which is supported by results observed from electron‐activated dissociation techniques. Therefore, the free radical activated glycan sequencing (FRAGS) reagent was developed so as to possess the characteristics of both acid‐base and free radical chemistry, thus generating information‐rich glycosidic bond and cross‐ring cleavages. Alternatively, the free radical processes can be induced via photodissociation of the specific carbon‐iodine bond which gives birth to similar fragmentation patterns as the FRAGS reagent. Furthermore, the methylated‐FRAGS (Me‐FRAGS) reagent was developed to eliminate glycan rearrangements by way of a fixed charged as opposed to a labile proton, which would otherwise yield additional, yet unpredictable, fragmentations including internal residue losses or multiple external residue losses. Lastly, to further enhance glycan enrichment and characterization, solid‐support FRAGS was developed.

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

Native top-down proteomics allows for both proteoform identification and high-order structure characteri-zation for cellular protein complexes. Unfortunately, tandem MS-based fragmentation efficiencies for such targets are low due to an increase in analyte ion mass and the low ion charge states that characterize native MS data. Multiple fragmenta-tion methods can be integrated in order to increase protein complex sequence coverage, but this typically requires use of specialized hardware and software. Free-radical initiated peptide sequencing (FRIPS) enables access to charge-remote and electron-based fragmentation channels within the context of conventional CID experiments. Here, we optimize FRIPS la-belling for native top-down sequencing experiments. Our labelling approach is able to access intact complexes with TEMPO-based FRIPS reagents without significant protein denaturation or assembly disruption. By combining CID and FRIPS datasets, we observed sequence coverage improvements as large as 50% for protein complexes ranging from 36 kDa to 106 kDa. Fragment ion production in these experiments was increased by as much as 102%. In general, our results indicate that TEMPO-based FRIPS reagents have the potential to dramatically increase sequence coverage obtained in na-tive top-down experiments.