Screen-printed digital microfluidics combined with surface acoustic wave nebulization for hydrogen-deuterium exchange measurements.

An inexpensive digital microfluidic (DMF) chip was fabricated by screen-printing electrodes on a sheet of polyimide. This device was manually integrated with surface acoustic wave nebulization (SAWN) MS to conduct hydrogen/deuterium exchange (HDX) of peptides. The HDX experiment was performed by DMF mixing of one aqueous droplet of angiotensin II with a second containing various concentrations of D2O. Subsequently, the degree of HDX was measured immediately by SAWN-MS. As expected for a small peptide, the isotopically resolved mass spectrum for angiotensin revealed that maximum deuterium exchange was achieved using 50% D2O. Additionally, using SAWN-MS alone, the global HDX kinetics of ubiquitin were found to be similar to published NMR data and back exchange rates for the uncooled apparatus using high inlet capillary temperatures was less than 6%.

Dissecting the dynamic conformations of the metamorphic protein lymphotactin.

A mass spectrometer provides an ideal laboratory to probe the structure and stability of isolated protein ions. Interrogation of each discrete mass/charge-separated species enables the determination of the intrinsic stability of a protein fold, gaining snapshots of unfolding pathways. In solution, the metamorphic protein lymphotactin (Ltn) exists in equilibrium between two distinct conformations, a monomeric (Ltn10) and a dimeric (Ltn40) fold. Here, we use electron capture dissociation (ECD) and drift tube ion mobility-mass spectrometry (DT IM-MS) to analyze both forms and use molecular dynamics (MD) to consider how the solution fold alters in a solvent-free environment. DT IM-MS reveals significant conformational flexibility for the monomer, while the dimer appears more conformationally restricted. These findings are supported by MD calculations, which reveal how salt bridges stabilize the conformers in vacuo. Following ECD experiments, a distinctive fragmentation pattern is obtained for both the monomer and dimer. Monomer fragmentation becomes more pronounced with increasing charge state especially in the disordered regions and C-terminal α-helix in the solution fold. Lower levels of fragmentation are seen in the ß-sheet regions and in regions that contain salt bridges, identified by MD simulations. The lowest charge state of the dimer for which we obtain ECD data ([D+9H](9+)) exhibits extensive fragmentation with no relationship to the solution fold and has a smaller collision cross section (CCS) than charge states 10-13+, suggesting a "collapsed" encounter complex. Other charge states of the dimer, as for the monomer, are resistant to fragmentation in regions of ß-sheets in the solution fold. This study provides evidence for preservation and loss of global fold and secondary structural elements, providing a tantalizing glimpse into the power of the emerging field of native top-down mass spectrometry.

Mass spectrometry imaging for dissecting steroid intracrinology within target tissues.

Steroid concentrations within tissues are modulated by intracellular enzymes. Such "steroid intracrinology" influences hormone-dependent cancers and obesity and provides targets for pharmacological inhibition. However, no high resolution methods exist to quantify steroids within target tissues. We developed mass spectrometry imaging (MSI), combining matrix assisted laser desorption ionization with on-tissue derivatization with Girard T and Fourier transform ion cyclotron resonance mass spectrometry, to quantify substrate and product (11-dehydrocorticosterone and corticosterone) of the glucocorticoid-amplifying enzyme 11ß-HSD1. Regional steroid distribution was imaged at 150-200 µm resolution in rat adrenal gland and mouse brain sections and confirmed with collision induced dissociation/liquid extraction surface analysis. In brains of mice with 11ß-HSD1 deficiency or inhibition, MSI quantified changes in subregional corticosterone/11-dehydrocorticosterone ratio, distribution of inhibitor, and accumulation of the alternative 11ß-HSD1 substrate, 7-ketocholesterol. MSI data correlated well with LC-MS/MS in whole brain homogenates. MSI with derivatization is a powerful new tool to investigate steroid biology within tissues.

Appropriate degree of trust: deriving confidence metrics for automatic peak assignment in high-resolution mass spectrometry.

Techniques for deriving confidence metrics for the reliability of automatically assigned elemental formulas in complex spectra, from high-resolution mass spectrometers, are described. These metrics can help an analyst to place an appropriate degree of trust in the results obtained from automated spectral analysis of, for example, natural organic materials. To provide these metrics of confidence, common mass spectrometric tests for reliability of peak assignment (mass accuracy/error, relative ion abundance, and rings-plus-double-bonds equivalence) are combined with novel confidence metrics based on the interconnectivity and consistency of a mass difference or mass defect based peak inference network and on the confidence of the initial library matches. These are shown to provide improved peak assignment confidence over manual or simple automatic assignment methods.

Use of an artificial immune system derived method for the charge state assignment of small-molecule mass spectra.

Knowing the charge state of an ion in a mass spectrum is crucial to being able to assign a formula to it. For many small-molecule peaks in complex mass spectra, the intensities of the isotopic peaks are too low to allow the charge state to be calculated from isotopic spacings, which is the basis of the conventional method of determining the charge state of an ion. A novel artificial intelligence derived method for identifying the charge state of ions, in the absence of any isotopic information or a series of charge states, has been developed using an artificial immune system approach. This technique has been tested against synthetic and real data sets and has proven successful in identifying the majority of multiply charged ions, thereby significantly improving the peak assignment rate and confidence.

Precursor ion independent algorithm for top-down shotgun proteomics.

We present a precursor ion independent top-down algorithm (PIITA) for use in automated assignment of protein identifications from tandem mass spectra of whole proteins. To acquire the data, we utilize data-dependent acquisition to select protein precursor ions eluting from a C4-based HPLC column for collision induced dissociation in the linear ion trap of an LTQ-Orbitrap mass spectrometer. Gas-phase fractionation is used to increase the number of acquired tandem mass spectra, all of which are recorded in the Orbitrap mass analyzer. To identify proteins, the PIITA algorithm compares deconvoluted, deisotoped, observed tandem mass spectra to all possible theoretical tandem mass spectra for each protein in a genomic sequence database without regard for measured parent ion mass. Only after a protein is identified, is any difference in measured and theoretical precursor mass used to identify and locate post-translation modifications. We demonstrate the application of PIITA to data generated via our wet-lab approach on a Salmonella typhimurium outer membrane extract and compare these results to bottom-up analysis. From these data, we identify 154 proteins by top-down analysis, 73 of which were not identified in a parallel bottom-up analysis. We also identify 201 unique isoforms of these 154 proteins at a false discovery rate (FDR) of <1%.

Destabilizing missense mutations in the tumour suppressor protein p53 enhance its ubiquitination in vitro and in vivo.

p53 ubiquitination catalysed by MDM2 (murine double minute clone 2 oncoprotein) provides a biochemical assay to dissect stages in E3-ubiquitin-ligase-catalysed ubiquitination of a conformationally flexible protein. A mutant form of p53 (p53(F270A)) containing a mutation in the second MDM2-docking site in the DNA-binding domain of p53 (F270A) is susceptible to modification of long-lived and high-molecular-mass covalent adducts in vivo. Mutant F270A is hyperubiquitinated in cells as defined by immunoprecipitation and immunoblotting with an anti-ubiquitin antibody. Transfection of His-tagged ubiquitin along with p53(R175H) or p53(F270A) also results in selective hyperubiquitination in cells under conditions where wild-type p53 is refractory to covalent modification. The extent of mutant p53(R175H) or p53(F270A) unfolding in cells as defined by exposure of the DO-12 epitope correlates with the extent of hyperubiquitination, suggesting a link between substrate conformation and E3 ligase function. The p53(F270A:6KR) chimaeric mutant (where 6KR refers to the simultaneous mutation of lysine residues at positions 370, 372, 373, 381, 382 and 386 to arginine) maintains the high-molecular-mass covalent adducts and is modified in an MDM2-dependent manner. Using an in vitro ubiquitination system, mutant p53(F270A) and the p53(F270A:6KR) chimaeric mutant is also subject to hyperubiquitination outwith the C-terminal domain, indicating direct recognition of the mutant p53 conformation by (a) factor(s) in the cell-free ubiquitination system. These data identify an in vitro and in vivo assay with which to dissect how oligomeric protein conformational alterations are linked to substrate ubiquitination in cells. This has implications for understanding the recognition of misfolded proteins during aging and in human diseases such as cancer.

Analysis of low oxidation state transition metal clusters by laser desorption/ionization time-of-flight mass spectrometry.

A variety of homonuclear and heteronuclear transition metal carbonyl clusters have been analyzed by ultraviolet laser desorption/ionization time-of-flight mass spectrometry. The spectra were recorded in negative and positive ion modes, using both linear and reflective techniques. A range of different clusters based on different nuclearities, geometries, and ligand types, which include hydrides, phosphines, nitriles, and cyclopentadienyl ligands and naked main group atoms, were studied. These experiments have allowed us to construct a detailed picture of the technique for the analysis of transition metal carbonyl clusters and their derivatives. In general, extensive reactions are observed, cluster aggregation reactions in particular, and from a comparison of the spectra obtained, some mechanistic inferences concerning the aggregation processes have been drawn.