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Top-Down Analysis of Proteins in Low Charge States

  • Aarti Bashyal
  • James D. Sanders
  • Dustin D. Holden
  • Jennifer S. BrodbeltEmail author
Research Article

Abstract

The impact of charging methods on the dissociation behavior of intact proteins in low charge states is investigated using HCD and 193 nm UVPD. Low charge states are produced for seven different proteins using the following four different methods: (1) proton transfer reactions of ions in high charge states generated from conventional denaturing solutions; (2) ESI of proteins in solutions of high ionic strength to enhance retention of folded native-like conformations; (3) ESI of proteins in high pH solutions to limit protonation; and (4) ESI of carbamylated proteins. Comparison of sequence coverages, degree of preferential cleavages, and types and distribution of fragment ions reveals a number of differences in the fragmentation patterns depending on the method used to generate the ions. More notable differences in these metrics are observed upon HCD than upon UVPD. The fragmentation caused by HCD is influenced more significantly by the presence/absence of mobile protons, a factor that modulates the degree of preferential cleavages and net sequence coverages. Carbamylation of the lysines and the N-terminus of the proteins alters the proton mobility by reducing the number of proton-sequestering, highly basic sites as evidenced by decreased preferential fragmentation C-terminal to Asp or N-terminal to Pro upon HCD. UVPD is less dependent on the method used to generate the low charge states and favors non-specific fragmentation, an outcome which is important for obtaining high sequence coverage of intact proteins.

Keywords

Top-down Ultraviolet photodissociation Protein Low charge state 

Notes

Acknowledgements

We appreciate the donation of proteins from Dr. Kevin Dalby (calmodulin) and Dr. Lauren Webb (Staph nuclease). Funding from the NIH (R01GM121714) and the Robert A. Welch Foundation (F-1155) is acknowledged.

Supplementary material

13361_2019_2146_MOESM1_ESM.pdf (2.5 mb)
ESM 1 (PDF 2605 kb)

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Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of ChemistryThe University of Texas at AustinAustinUSA

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