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

Version 1

      Publication Details (including relevant citation   information):

      J Struct Funct Genomics (2005) 6: 129-134.

      Gao X, Bain K, Bonanno JB, Buchanan M, Henderson D, Lorimer D,   Marsh C, Reynes JA, Sauder JM, Schwinn K, Thai C, Burley SK


      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.

      Address (URL): http://www.ncbi.nlm.nih.gov/pubmed/16211509