Development of Novel Free Radical Initiated Peptide Sequencing Reagent: Application to Identification and Characterization of Peptides by Mass Spectrometry

Abstract

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,

Free Radical-based Sequencing for Native Top-Down Mass Spectrometry

“…Both 2,5-dioxopyrrolidin-1-yl 2-((2,2,6,6-tetramethylpiperidin-1-yl)oxy)acetate Nhydroxysuccinimide ester (aTEMPO) and 2,5-dioxopyrrolidin-1-yl 5-(((2,2,6,6-tetramethylpiperidin-1-yl)oxy)methyl)nicotinate N-hydroxysuccinimide ester (pTEMPO) were synthesized as previously published with no further modifications. 35,38 At pH 8, the reagents target Lys residues and the n-terminus. 39,40 Ubiquitin Labelling Previous TEMPO labelling workflows have been carried out under denaturing conditions.…”

“…36 In this work, we seek to extend FRIPS approaches for nTD methods. While several such tagging reagents have been developed, 29 our efforts have focused on TEMPO-based FRIPS reagents, 37,38 due to their efficient n-terminal and lysine residue labelling, low radicalization energy, 37 and their ability to both radicalize and fragment in one-step within Q-IM-TOF instruments. 31 Typical TEMPO labeling protocols involve reaction conditions that promote protein denaturation, and as such we have screened a number of TEMPO tags as well as developed modified reaction conditions that optimally preserve native protein structures for FRIPS nTD experiments.…”

Resin and Magnetic Nanoparticle-Based Free Radical Probes for Glycan Capture, Isolation, and Structural Characterization

“…Recently, free radical chemistry 46 has regained great attention in the field of biomolecular characterization. [47][48][49][50][51] Inspired by the achievement of electron activated dissociation (ExD) for glycan structure analysis, which usually involve fragmentation via low energy free radical dissociation pathways, we recently developed a novel free radical activation glycan sequencing reagent (FRAGS), along with a methylated free radical activated glycan sequencing reagent (Me-FRAGS) for glycan structural characterization. 26,52 Free radical-directed systematic glycan dissociation with high fragmentation efficiency has been obtained using these two free radical reagents.…”

“…However, such approaches require special instrumentation to allow for interaction of an electron source with targeted ions. Recently, free radical chemistry has regained great attention in the field of biomolecular characterization. − …”

Free-Radical-Mediated Glycan Isomer Differentiation

“…Free-radical-driven dissociation techniques, including radical-directed dissociation (RDD), − electron capture dissociation (ECD), ,− electron transfer dissociation (ETD), ,− electron detachment dissociation (EDD), , and electronic excitation dissociation ,− (EED), have shown especially great promise for glycan structural characterization. In addition, free radical chemistry has also attracted significant attention in the field of proteomics − and lipidomics. − Although various mass spectrometric dissociation techniques have been applied to glycan structural elucidation, it is still challenging to differentiate stereoisomers, especially anomers and epimers. Tandem mass spectrometry, , ion-mobility mass spectrometry, UVPD, and EDD have been developed for the differentiation of glycan isomers.…”