High-throughput limited proteolysis/mass spectrometry for protein domain elucidation

Xia Gao, Kevin Bain, Jeffery B. Bonanno, Michelle Buchanan, Davin Henderson, Don Lorimer, Curtis Marsh, Julie A. Reynes, J. Michael Sauder, Ken Schwinn, Chau Thai, Stephen K. Burley

Research output: Contribution to journalArticle

45 Scopus citations

Abstract

High-resolution structural information is important for improving our understanding of protein function in vitro and in vivo and providing information to enable drug discovery. The process leading to X-ray structure determination is often time consuming and labor intensive. It requires informed decisions in expression construct design, expression host selection, and strategies for protein purification, crystallization and structure determination. Previously published studies have demonstrated that compact globular domains defined by limited proteolysis represent good candidates for production of diffraction quality crystals [1-7]. Integration of mass spectrometry and proteolysis experiments can provide accurate definition of domain boundaries at unprecedented rates. We have conducted a critical evaluation of this approach with 400 target proteins produced by SGX (Structural GenomiX, Inc.) for the New York Structural GenomiX Research Consortium (NYSGXRC; http://www.nysgxrc.org ) under the auspices of the National Institute of General Medical Sciences Protein Structure Initiative ( http://www.nigms.nih.gov/psi ). The objectives of this study were to develop parallel/automated protocols for proteolytic digestion and data acquisition for multiple proteins, and to carry out a systematic study to correlate domain definition via proteolysis with outcomes of crystallization and structure determination attempts. Initial results from this work demonstrate that proteins yielding diffraction quality crystals are typically resistant to proteolysis. Large-scale sub cloning and subsequent testing of expression, solubility, and crystallizability of proteolytically defined truncations is currently underway.

Original languageEnglish (US)
Pages (from-to)129-134
Number of pages6
JournalJournal of Structural and Functional Genomics
Volume6
Issue number2-3
DOIs
StatePublished - Sep 1 2005
Externally publishedYes

    Fingerprint

Keywords

  • Automation
  • Domain definition
  • LPMS
  • Limited proteolysis
  • Mass spectrometry
  • Protein domain

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Genetics

Cite this

Gao, X., Bain, K., Bonanno, J. B., Buchanan, M., Henderson, D., Lorimer, D., Marsh, C., Reynes, J. A., Sauder, J. M., Schwinn, K., Thai, C., & Burley, S. K. (2005). High-throughput limited proteolysis/mass spectrometry for protein domain elucidation. Journal of Structural and Functional Genomics, 6(2-3), 129-134. https://doi.org/10.1007/s10969-005-1918-5