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Studies of Three-Dimensional Protein Structure using Mass Spectrometry (MS3D)

presented by

Gary H. Kruppa
Bruker Daltonics Inc. and Sandia National Lab.

July 22, 2004

The Scripps Research Institute, W.M. Keck Foundation Amphitheater


Background:

Dr. Kruppa has been involved with ICR since 1980, when he began doing research as an undergraduate in the laboratory of Prof. Douglas Ridge at the University of Delaware. From 1991 to 2001 he worked at Bruker Daltonics in the FTMS group, eventually becoming Vice President for FTMS. In 2001 he left Bruker to pursue a research program in the study of protein structure by mass spectrometry at Sandia National Labs in Livermore, CA. In 2003 he re-joined Bruker Daltonics as Vice President for Western Region Operations, while maintaining a visiting scientist relationship with Sandia National Labs. He has co-authored more than 30 publications and two patents.

Abstract:

High throughput protein structure determination efforts continue to rely on multidimensional NMR and X-Ray crystallographic methods. These methods provide detailed, high-resolution protein structures. However, X-Ray crystallography requires single crystals of a protein, which are sometimes difficult or impossible to grow. NMR requires milligrams of protein, which must be soluble in an NMR compatible solvent system, and NMR investigations are currently limited to proteins of approximately 30 kDa or less. Given these and other difficulties, several groups have been investigating the use of ?sparse? or ?minimal? constraint sets, i.e. constraint sets obtained from NMR, EPR, FRET, X-Ray crystallography, or some combination of these methods which contain less than the number of distance constraints expected from an ideal multi-dimensional NMR or X-Ray crystallographic data set. The use of experimental constraints from a minimal constraint experiment has been shown to be a valuable way to speed protein structure determination using NMR. Intra-molecular chemical cross-linking followed by mass spectrometric analysis has recently been shown to have potential as a new method for obtaining distance constraints between reactive side-chains in protein, and named MS3D. The advantages of the MS3D method for obtaining distance constraints include the ability to work with small amounts of protein (10 mg or less), no need for single crystals, and the solvent system is limited only by the cross-linking chemistry. The development of an automated, sensitive method using MS3D would clearly have a great impact on high throughput structure determination efforts. This impact would be due to several new capabilities provided by the MS3D method: The ability to pre-screen proteins for fold family at low expression levels, before scale-up of the expression to obtain sufficient protein for NMR or X-Ray analysis; Analysis of targets that prove recalcitrant to NMR or X-Ray methods; Speeding and improving structure determination from sparse NMR data. Despite the initial report indicating the potential of MS3D, the number of distance constraints reported in the literature using this method has been limited. Several publications have indicated that the cross-linking chemistry itself may be difficult to optimize, and the method is further complicated by the difficulty of using proteolytic digestion and HPLC-MS to localize the cross-links. We have developed a whole protein approach (known as the top-down method) using Fourier Transform Mass Spectrometry (FTMS) to localize the cross-links using MSn analysis rather than proteolytic digestions. This method also facilitates optimization of the cross-linking chemistries. We will present results on the study of the structure of ubiquitin and residue specific reactivity in ubiquitin using this top down approach, and discuss the extension of the method to other proteins.

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