Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry
- Nicole A. HaverlandNicole A. HaverlandDepartment of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, 60208, Evanston, IL, USAMore by Nicole A. Haverland,
- Owen S. SkinnerOwen S. SkinnerDepartment of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, 60208, Evanston, IL, USAMore by Owen S. Skinner,
- Ryan T. FellersRyan T. FellersDepartment of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, 60208, Evanston, IL, USAMore by Ryan T. Fellers,
- Areeba A. TariqAreeba A. TariqDepartment of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, 60208, Evanston, IL, USAMore by Areeba A. Tariq,
- Bryan P. EarlyBryan P. EarlyDepartment of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, 60208, Evanston, IL, USAMore by Bryan P. Early,
- Richard D. LeDucRichard D. LeDucDepartment of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, 60208, Evanston, IL, USAMore by Richard D. LeDuc,
- Luca FornelliLuca FornelliDepartment of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, 60208, Evanston, IL, USAMore by Luca Fornelli,
- Philip D. ComptonPhilip D. ComptonDepartment of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, 60208, Evanston, IL, USAMore by Philip D. Compton, and
- Neil L. KelleherNeil L. KelleherDepartment of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, 60208, Evanston, IL, USAMore by Neil L. Kelleher
Abstract
Fragmentation of intact proteins in the gas phase is influenced by amino acid composition, the mass and charge of precursor ions, higher order structure, and the dissociation technique used. The likelihood of fragmentation occurring between a pair of residues is referred to as the fragmentation propensity and is calculated by dividing the total number of assigned fragmentation events by the total number of possible fragmentation events for each residue pair. Here, we describe general fragmentation propensities when performing top-down mass spectrometry (TDMS) using denaturing or native electrospray ionization. A total of 5311 matched fragmentation sites were collected for 131 proteoforms that were analyzed over 165 experiments using native top-down mass spectrometry (nTDMS). These data were used to determine the fragmentation propensities for 399 residue pairs. In comparison to denatured top-down mass spectrometry (dTDMS), the fragmentation pathways occurring either N-terminal to proline or C-terminal to aspartic acid were even more enhanced in nTDMS compared with other residues. More generally, 257/399 (64%) of the fragmentation propensities were significantly altered (P ≤ 0.05) when using nTDMS compared with dTDMS, and of these, 123 were altered by 2-fold or greater. The most notable enhancements of fragmentation propensities for TDMS in native versus denatured mode occurred (1) C-terminal to aspartic acid, (2) between phenylalanine and tryptophan (F|W), and (3) between tryptophan and alanine (W|A). The fragmentation propensities presented here will be of high value in the development of tailored scoring systems used in nTDMS of both intact proteins and protein complexes.
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