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

  • Kaylee Gaspar
  • Kimberly Fabijanczuk
  • Tara Otegui
  • Jose Acosta
  • Jinshan GaoEmail author
Research Article


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) N-hydroxysuccinimide (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 Abstract


Free radical Peptide sequencing Hydrophobic peptides Peptides without basic amino acid residues Charge localize Insulin 



This work is supported by the National Institutes of Health through grant 1R15GM121986-01A1 and National Science Foundation through grant CHEM1709272.

Supplementary material

13361_2018_2114_MOESM1_ESM.docx (1.7 mb)
ESM 1 (DOCX 1742 kb)


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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Department of Chemistry and Biochemistry, Center for Quantitative Obesity ResearchMontclair State UniversityMontclairUSA

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