Maximizing Sequence Coverage in Top-Down Proteomics by Automated Multimodal Gas-Phase Protein Fragmentation

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

  • Pavel V. Shliaha, Southern Denmark University
  • ,
  • Sebastian Gibb, University Medicine Greifswald
  • ,
  • Vladimir Gorshkov, Southern Denmark University
  • ,
  • Malena Schack Jespersen, Southern Denmark University, Danmark
  • Gregers R. Andersen
  • Derek Bailey, Thermo Fisher Scientific
  • ,
  • Jacob Schwartz, Thermo Fisher Scientific
  • ,
  • Shannon Eliuk, Thermo Fisher Scientific
  • ,
  • Veit Schwämmle, Southern Denmark University
  • ,
  • Ole N. Jensen, Southern Denmark University

Intact protein sequencing by tandem mass spectrometry (MS/MS), known as top-down protein sequencing, relies on efficient gas-phase fragmentation at multiple experimental conditions to achieve extensive amino acid sequence coverage. We developed the "topdownr" R-package for automated construction of multimodal (i.e., involving CID, HCD, ETD, ETciD, EThcD, and UVPD) MS/MS fragmentation methods on an orbitrap instrument platform and systematic analysis of the resultant spectra. We used topdownr to generate and analyze thousands of MS/MS spectra for five intact proteins of 10-30 kDa. We achieved 90-100% coverage for the proteins tested and derived guiding principles for efficient sequencing of intact proteins. The data analysis workflow and statistical models of topdownr software and multimodal MS/MS experiments provide a framework for optimizing MS/MS sequencing for any intact protein. Refined topdownr software will be suited for comprehensive characterization of protein pharmaceuticals and eventually also for de novo sequencing and detailed characterization of intact proteins.

OriginalsprogEngelsk
TidsskriftAnalytical Chemistry
Vol/bind90
Nummer21
Sider (fra-til)12519-12526
ISSN0003-2700
DOI
StatusUdgivet - 2018

Se relationer på Aarhus Universitet Citationsformater

ID: 134657648