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

Abstract

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.

Discerning ambiguous internal fragment assignments that are generated in top-down mass spectrometry with cyclic ion-mobility enhances protein sequence information

“…In light of this issue, internal fragments (fragment ions containing neither the N nor the C terminus) have recently been incorporated into TD-MS data analysis. [13][14][15][16][17][18] The addition of internal fragments in TD-MS analysis has been shown to enhance protein sequence information, with many reports showing near complete protein sequence coverage for a range of common test proteins. 14 Further, Wei and co-workers demonstrated that the inclusion of internal fragments can be utilised to gain extensive protein structure characterisation by enabling enhanced determination of disulfide bond connectivity.…”