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 ug 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.

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, before the invention of FT(ICR)MS.  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.

Meeting details

Date:	Wednesday	March 3, 2004
Time:	6:00 pm	Social hour, registration (no-host cocktails)
	7:00 pm	Dinner
	8:00 pm	Lecture
Dinner:	Buffet featuring:	Grilled Chicken Breast Toscano
		Stuffed Petrale Sole
		Penne ala Dominic
	includes	various side dishes & dessert
Cost:	$25.00	BAMS members.  Reservations required by noon on Friday February 27,2004
	$35.00	Non-members.  Reservations required by noon on Friday February 27,2004
	$15.00	Students only.  Reservations required by noon on Friday February 27,2004

Note: 2004 dues need to be paid to obtain member price.  Dues ($20) may be paid while registering for dinner.

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Dominic's at Oyster Point
360 Oyster Point Blvd.
South San Francisco, California 94080
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The San Francisco Bay Area Mass Spectrometry discussion group was formed in 1980 to provide a regular gathering for people interested in mass spectrometry and allied topics. BAMS currently has a membership of about 280 individual and 20 corporate members, and meets 8-10 times per year for a midweek dinner and lecture.  Meetings attract between 30 and 90 people, and are held at a restaurant or hotel in the bay area convenient for our speaker.  We usually convene at 6:00 pm for cocktails, dinner at 7:00 pm, and lecture at 8:15 pm.

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Last update: 2/20/2004