Mass Spectrometry Guided In Situ Proteolysis to Obtain Crystals for X-ray Structure Determination

Journal Article · Mon Apr 30 04:00:00 EDT 2012 · J. Am. Soc. Mass. Spectrom.

DOI:https://doi.org/10.1016/j.jasms.2010.06.015· OSTI ID:1037491

Gheyi, Tarun; Rodgers, Logan; Romero, Richard; Sauder, J Michael; Burley, Stephen K ^[1]

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1. Lilly

A strategy for increasing the efficiency of protein crystallization/structure determination with mass spectrometry has been developed. This approach combines insights from limited proteolysis/mass spectrometry and crystallization via in situ proteolysis. The procedure seeks to identify protease-resistant polypeptide chain segments from purified proteins on the time-scale of crystal formation, and subsequently crystallizing the target protein in the presence of the optimal protease at the right relative concentration. We report our experience with 10 proteins of unknown structure, two of which yielded high-resolution X-ray structures. The advantage of this approach comes from its ability to select only those structure determination candidates that are likely to benefit from application of in situ proteolysis, using conditions most likely to result in formation of a stable proteolytic digestion product suitable for crystallization.

• Research Organization: Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
• Sponsoring Organization: NIH
• OSTI ID: 1037491
• Journal Information: J. Am. Soc. Mass. Spectrom., Journal Name: J. Am. Soc. Mass. Spectrom. Journal Issue: 10, 2010 Vol. 10; ISSN 1044-0305
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
CRYSTALLIZATION
DIGESTION
EFFICIENCY
MASS SPECTROSCOPY
POLYPEPTIDES
PROTEINS
PROTEOLYSIS
SPECTROSCOPY
TARGETS