High-throughput Limited Proteolysis/Mass Spectrometry for Protein Domain Elucidation

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.

Time-resolved limited proteolysis of mitogen-activated protein kinase-activated protein kinase-2 determined by LC/MS only

“…We have found that if a clear mass spectrum was obtained on an intact target protein, its proteolytic fragments were also measured reliably and the current method was applicable. Because this method identifies flexible and stable regions of proteins, it is useful in the design of protein constructs for X-ray crystallography studies [29], as well as for studying proteins with unknown structures. It is also conceivable that when a high-resolution mass spectrometer such as Fourier transform mass spectrometer (FTMS) is used, this method can be applied to much larger proteins, since the corresponding mass window for selecting potential subsequences can be significantly reduced for FTMS.…”

N- and C-Terminal Truncations to Enhance Protein Solubility and Crystallization: Predicting Protein Domain Boundaries with Bioinformatics Tools

“…If full length protein is available, limited proteolysis combined with mass spectrometric (MS) approaches can determine core folded domains, as connecting unfolded sequence or disordered termini may be trimmed away by proteases, with core domains identified by MS [40]. In addition, the advent of powerful high-throughput screening of random or combinatorial protein truncation or mutation libraries allows an unbiased approach with no prior knowledge required [41].…”

Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting

“…against protein domains of known structure), disorder predictions, and secondary structure predictions (Mooij et al, 2009). In silico analysis of the target can be complemented by experimental methods, such as partial proteolysis (Gao et al, 2005).…”