Activation of AMP-activated protein kinase revealed by hydrogen/deuterium exchange mass spectrometry.

AMP-activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme with hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin, and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and ß subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the ß subunit and in the kinase domain of the α subunit, suggesting that the molecular binding site of the latter resides between the α and ß subunits. The distinct short- and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands.

HDX workbench: software for the analysis of H/D exchange MS data.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established method for the interrogation of protein conformation and dynamics. While the data analysis challenge of HDX-MS has been addressed by a number of software packages, new computational tools are needed to keep pace with the improved methods and throughput of this technique. To address these needs, we report an integrated desktop program titled HDX Workbench, which facilitates automation, management, visualization, and statistical cross-comparison of large HDX data sets. Using the software, validated data analysis can be achieved at the rate of generation. The application is available at the project home page http://hdx.florida.scripps.edu .

Automated hydrogen/deuterium exchange electron transfer dissociation high resolution mass spectrometry measured at single-amide resolution.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a well established method for the measurement of solution-phase deuterium incorporation into proteins, which can provide insight into protein conformational mobility. However, most HDX measurements are constrained to regions of the protein where pepsin proteolysis allows detection at peptide resolution. Recently, single-amide resolution deuterium incorporation has been achieved by limiting gas-phase scrambling in the mass spectrometer. This was accomplished by employing a combination of soft ionization and desolvation conditions coupled with the radical-driven fragmentation technique electron transfer dissociation (ETD). Here, a hybrid LTQ-Orbitrap XL is systematically evaluated for its utility in providing single-amide deuterium incorporation for differential HDX analysis of a nuclear receptor upon binding small molecule ligands. We are able to show that instrumental parameters can be optimized to minimize scrambling and can be incorporated into an established and fully automated HDX platform making differential single-amide HDX possible for bottom-up analysis of complex systems. We have applied this system to determine differential single amide resolution HDX data for the peroxizome proliferator activated receptor bound with two ligands of interest.

Considerations for quantification of lipids in nerve tissue using matrix-assisted laser desorption/ionization mass spectrometric imaging.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging is a technique that provides the ability to identify and characterize endogenous and exogenous compounds spatially within tissue with relatively little sample preparation. While it is a proven methodology for qualitative analysis, little has been reported for its utility in quantitative measurements. In the current work, inherent challenges in MALDI quantification are addressed. Signal response is monitored over successive analyses of a single tissue section to minimize error due to variability in the laser, matrix application, and sample inhomogeneity. Methods for the application of an internal standard to tissue sections are evaluated and used to quantify endogenous lipids in nerve tissue. A precision of 5% or less standard error was achieved, illustrating that MALDI imaging offers a reliable means of in situ quantification for microgram-sized samples and requires minimal sample preparation.

Imaging of lipids in spinal cord using intermediate pressure matrix-assisted laser desorption-linear ion trap/Orbitrap MS.

A hybrid linear ion trap/Orbitrap mass spectrometer was used to perform tandem mass spectrometry (MS/MS) experiments and high-resolution mass analysis of lipids desorbed from nerve tissue. A dramatic improvement in mass spectral resolution and a decrease in background are observed in the spectra collected from the Orbitrap mass analyzer, which allows generation of more accurate mass spectrometric images of the distribution of lipids within nerve tissue. Employment of both mass analyzers provides a rapid and reliable means of compound identification based on MS/MS fragmentation and high-resolution mass spectrometry (HRMS) accurate mass.

MALDI-linear ion trap microprobe MS/MS studies of the effects of dichloroacetate on lipid content of nerve tissue.

The direct analysis of tissue from both the central and peripheral nervous systems of control rats and those administered the potential neurotoxin dichloroacetate (DCA) was investigated using an intermediate-pressure matrix-assisted laser desorption/ionization (IP-MALDI) source coupled to a linear ion trap (LIT) mass spectrometer. The matrix, 2,5-dihydroxybenzoic acid, was applied to the tissue using a novel automated inkjet printer system. The MSn capabilities of the LIT allowed identification of lipids desorbed directly from tissue. A marked decrease is observed in the intensity of lipid ions in spinal cord and sciatic nerve tissues from rats exposed to DCA. The results also demonstrate the rapid, sensitive, and semiquantitative capabilities of this method.