Isotopically Labeled Free Radical Initiated Peptide Sequencing (FRIPS) Tags for Integrated Protein Quantification and Characterization

Proteins are essential biomolecules with diverse roles in structure, transport, and enzymatic catalysis. Oftentimes, proteins serve as biomarkers for disease diagnosis and progression, making quantification and characterization of proteins highly important. Mass spectrometry (MS) is commonly used in proteomics, with tandem MS enabling sequencing and structural analysis. Free radical initiated peptide sequencing (FRIPS) provides an alternative fragmentation strategy by attaching a radical-generating reagent to a peptide N-terminus. When activated, FRIPS drives radical-directed dissociation (RDD), which improves sequence coverage and identifies post-translational modifications that are often inaccessible by conventional MS methods.
Unlike collision-induced dissociation, which mainly produces b/y ions and is prone to neutral loss of labile PTMs, RDD initiates site-specific radical chemistry that yields c/z• and a/x fragments as well as side-chain cleavages. This mechanism reduces sequence bias, improves backbone coverage, preserves labile PTMs, and promotes cleavage of resistant linkages such as disulfides and cross-links.
A major limitation of FRIPS has been incompatibility with standard quantification methods like tandem mass tags (TMTs), which also target the peptide N-terminus. Building on the one-step FRIPS reagent, consisting of TEMPO, methyl benzyl succinic acid (Bz-Sc), and an N-hydroxysuccinimide ester reactive group, we synthesized two isotopically labeled isobaric FRIPS tags that function as TMT analogues. Fragmentation includes homolytic cleavage of TEMPO and the Bz-Sc-peptide bond, generating two products: the Bz-Sc-peptide radical, which initiates RDD and mimics the TMT mass balancer, and TEMPO•, which is oxidized to TEMPO+ and serves as the reporter ion for quantification. This bifunctional design enables simultaneous relative quantification and structural characterization, advancing proteomics by integrating two dimensions of analysis in one experiment.