Gary H. Kruppa

Automated Ambient Desorption−Ionization Platform for Surface Imaging Integrated with a Commercial Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

A fully automated atmospheric pressure ionization platform has been built and coupled with a commercial high-resolution Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) instrument. The outstanding performance of this instrument allowed screening on the basis of exact masses in imaging mode. The main novel aspect was in the integration of the atmospheric pressure ionization imaging into the current software for matrix-assisted laser desorption ionization (MALDI) imaging, which allows the user of this commercial dual-source mass spectrometer to perform MALDI-MS and different ambient MS imaging from the same user interface and to utilize the same software tools. Desorption electrospray ionization (DESI) and desorption atmospheric pressure photoionization (DAPPI) were chosen to test the ambient surface imaging capabilities of this new ionization platform. Results of DESI imaging experiments performed on brain tissue sections are in agreement with previous MS imaging reports obtained by DESI imaging, but due to the high resolution and mass accuracy of the FTICR instrument it was possible to resolve several ions at the same nominal mass in the DESI-MS spectra of brain tissue. These isobaric interferences at low resolution are due to the overlap of ions from different lipid classes with different biological relevance. It was demonstrated that with the use of high-resolution MS fast imaging screening of lipids can be achieved without any preseparation steps. DAPPI, which is a relatively new and less developed ambient ionization technique compared to DESI, was used in imaging mode for the first time ever. It showed promise in imaging of phytocompounds from plant leaves, and selective ionization of a sterol lipid was achieved by DAPPI from a brain tissue sample.

Capillary Electrophoresis-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for the Identification of Cationic Metabolites via a pH-Mediated Stacking-Transient Isotachophoretic Method

Capillary electrophoresis-mass spectrometry (CE-MS) is still widely regarded as an emerging tool in the field of metabolomics and metabolite profiling. A major reason for this is a reported lack of sensitivity of CE-MS when compared to gas chromatography-mass spectrometry GC/MS and liquid chromatography-mass spectrometry. The problems caused by the lack of sensitivity are exacerbated when CE is coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), due to the relatively low data acquisition rate of FT-ICR MS. Here, we demonstrate the use of an online CE sample preconcentration method that uses a combination of pH-mediated stacking and transient isotachophoresis, coupled with FT-ICR MS to improve the overall detection of cationic metabolites in the bacterium Desulfovibrio vulgaris Hildenborough. This method showed a significant increase in signal-to-noise ratio when compared to CE normal sample stacking, while providing good separation efficiency, reproducibility, and linearity. Detection limits for selected amino acids were between 0.1 and 2 microM. Furthermore, FT-ICR MS detection consistently demonstrated good mass resolution and sub-ppm mass accuracy.

Structure and dynamics of dark-state bovine rhodopsin revealed by chemical cross-linking and high-resolution mass spectrometry

Recent work using chemical cross‐linking to define interresidue distance constraints in proteins has shown that these constraints are useful for testing tertiary structural models. We applied this approach to the G‐protein‐coupled receptor bovine rhodopsin in its native membrane using lysine‐ and cysteine‐targeted bifunctional cross‐linking reagents. Cross‐linked proteolytic peptides of rhodopsin were identified by combined liquid chromatography and FT‐ICR mass spectrometry with automated data‐reduction and assignment software. Tandem mass spectrometry was used to verify cross‐link assignments and locate the exact sites of cross‐link attachment. Cross‐links were observed to form between 10 pairs of residues in dark‐state rhodopsin. For each pair, cross‐linkers with a range of linker lengths were tested to determine an experimental distance‐of‐closest‐approach (DCA) between reactive side‐chain atoms. In all, 28 cross‐links were identified using seven different cross‐linking reagents. Molecular mechanics procedures were applied to published crystal structure data to calculate energetically achievable theoretical DCAs between reactive atoms without altering the position of the protein backbone. Experimentally measured DCAs are generally in good agreement with the theoretical DCAs. However, a cross‐link between C316 and K325 in the C‐terminal region cannot be rationalized by DCA simulations and suggests that backbone reorientation relative to the crystal coordinates occurs on the timescale of cross‐linking reactions. Biochemical and spectroscopic data from other studies have found that the C‐terminal region is highly mobile in solution and not fully represented by X‐ray crystallography data. Our results show that chemical cross‐linking can provide reliable three‐dimensional structural information and insight into local conformational dynamics in a membrane protein.

A top-down approach to protein structure studies using chemical cross-linking and Fourier transform mass spectrometry

In a preliminary communication we described a top-down approach to the determination of chemical cross-link location in proteins using Fourier transform mass spectrometry (FT-MS). We have since extended the approach to use a series of homobifunctional crosslinkers with the same reactive functional groups, but different cross-linker arm lengths. Correlating cross-linking data across a series of related linkers allows the distance constraint derived from a cross-link between two reactive side chains to be determined more accurately and increases the confidence in the assignment of the cross-links. In ubiquitin, there are seven lysines with primary amino groups and the amino terminus. Disuccinimidyl suberate (DSS, cross-linker arm length = 11.4 Å), disuccinimidyl glutarate (DSG, cross-linker arm length = 7.5 Å) and disuccinimidyl tartrate (DST, cross-linker arm length = 5.8 Å) are homobifunctional cross-linking reagents that react specifically with primary amines. Using tandem mass spectrometry (MS/MS) on the singly, internally crosslinked precursor ion of ubiquitin, we found cross-links with DSS and DSG between the amino terminus and Lys 6, between Lys 6 and Lys 11, and between Lys 63 and Lys 48. Using disuccinimidyl tartrate (DST), the shortest cross-linker in the series, only the cross-links between the amino terminus and Lys 6, and between Lys 6 and Lys 11 were observed. The observed cross-links are consistent with the crystal structure of ubiquitin, if the lysine side chains and the amino terminus are assumed to have considerable flexibility. In a separate study, we probed the reactivity of the primary amino groups in ubiquitin using the amino acetylating reagent, N-hydroxy succinimidyl acetate (NHSAc), and a top-down approach to localize the acetylated lysine residues. The reactivity order obtained in that study (M1 ≈ K6 ≈ K48 ≈ K63) > K33 > K11 > (K27, K29), shows that the cross-link first formed in ubiquitin by reaction with DSS and DSG occurs between the most reactive residues.

Determination of phosphate position in hexose monosaccharides using an FTICR mass spectrometer: ion/molecule reactions, labeling studies, and dissociation mechanisms

Abstract Determination of phosphate position in carbohydrates using mass spectrometry is difficult due to the low energy loss of the phosphate either as a neutral or as an ion in MS/MS experiments. A possible solution to this problem is proposed in this work, whereby we use ion/molecule reactions in combination with tandem mass spectrometry to determine the site of phosphorylation on phosphorylated monosaccharides. Singly charged negative ions from phosphorylated monosaccharides are reacted with trimethyl borate in an FTICR MS analyzer cell to produce ion/molecule reaction products with the loss of a neutral methanol molecule. This reaction product likely involves a covalent bond between one of the phosphate oxygen atoms and boron. Derivatization of the phosphate in this manner allows stabilization of the phosphate group under SORI-CID conditions, allowing generation of ions characteristic of the phosphate linkage. Ion structures and dissociation mechanisms explaining these results are presented and discussed. The mechanistic studies suggest that the extra degrees of freedom provided by the 6-linked phosphate allows formation of diagnostic ions in the 6-linked case that are not formed from the 1-linked isomer. The dissociation of the ion/molecule reaction products using infrared multi-photon dissociation (IRMPD) as an activation method was also investigated. While SORI-CID and IRMPD activation yield similar dissociation patterns, the characteristic differences in the product ion spectra between the monosaccharides phosphorylated in the 1- and 6-positions are not observed using IRMPD.

The interfacial bioscience grand challenge.

This report is broken down into the following 3 sections: (1) Chemical Cross-linking and Mass Spectrometry Applied to Determination of Protein Structure and Dynamics; (2) Computational Modeling of Membrane Protein Structure and Dynamics; and (3) Studies of Toxin-Membrane Interactions using Single Molecule Biophysical Methods.