Grant: Research Tools and Instruments - Category 1 (<$150,000)

Support is requested for the acquisition of an "Advion Nanomate". This device is an essential accessory for our liquid chromatography mass spectrometry (LC/MS) system. We extensively use LC/MS methods for the analysis of protein digests in the context of research on biomolecular folding and dynamics. Unfortunately, our state-of-the-art MS equipment is completely overwhelmed by the numerous peptides that elute off the LC column in very narrow peaks. This is problematic because the outcome of our work hinges on a detailed tandem mass spectrometry (MS/MS) analysis of all these species. The current experiments suffer from poor sequence coverage since it is impossible to analyze all compounds of interest by on-line MS/MS. The NANOMATE solves this problem in an elegant fashion: Only part of the flow from the LC column is directed to the mass spectrometer, the other part is collected in small fractions. The fractions of interest are then robotically transferred into microtips, which are connected to a microfluidic nanoESI interface. The ultra-low flow rates used under nanospray conditions allow individual fractions to be infused for as much as one hour, such that every single peptide can be analyzed in detail. NanoESI has the added advantage that it is almost free of ion suppression. As a result, we will be able to carry out exhaustive peptide mapping with complete sequence coverage. A number of cutting-edge research projects depend critically on the availability of a NANOMATE: (A) Laser pump-probe experiments employing oxidative labeling will for the first time provide detailed structural information on the earliest events in protein folding. (B) Hydrogen/deuterium exchange studies will reveal whether enzymes are more dynamic during catalysis, or whether they function within the limitations given by the intrinsic dynamics of the resting state. (C) Monitoring protein-protein assembly processes requires nanoESI, since many noncovalent complexes dissociate under standard ESI conditions. (D) The NANOMATE will allow interfacing our mass spectrometer with novel custom-designed ultra-rapid mixing devices. Failure to secure funding for a NANOMATE would be a severe setback for our program.