David C. Muddiman

Performance characteristics of a new hybrid quadrupole time-of-flight tandem mass spectrometer (TripleTOF 5600).

The TripleTOF 5600 System, a hybrid quadrupole time-of-flight mass spectrometer, was evaluated to explore the key figures of merit in generating peptide and protein identifications that included spectral acquisition rates, data quality, proteome coverage, and biological depth. Employing a Saccharomyces cerevisiae tryptic digest, careful consideration of several performance features demonstrated that the speed of the TripleTOF contributed most to the resultant data. The TripleTOF system was operated with 8, 20, and 50 MS/MS events in an effort to compare with other MS technologies and to demonstrate the abilities of the instrument platform.

MSiReader: An Open-Source Interface to View and Analyze High Resolving Power MS Imaging Files on Matlab Platform

AbstractDuring the past decade, the field of mass spectrometry imaging (MSI) has greatly evolved, to a point where it has now been fully integrated by most vendors as an optional or dedicated platform that can be purchased with their instruments. However, the technology is not mature and multiple research groups in both academia and industry are still very actively studying the fundamentals of imaging techniques, adapting the technology to new ionization sources, and developing new applications. As a result, there important varieties of data file formats used to store mass spectrometry imaging data and, concurrent to the development of MSi, collaborative efforts have been undertaken to introduce common imaging data file formats. However, few free software packages to read and analyze files of these different formats are readily available. We introduce here MSiReader, a free open source application to read and analyze high resolution MSI data from the most common MSi data formats. The application is built on the Matlab platform (Mathworks, Natick, MA, USA) and includes a large selection of data analysis tools and features. People who are unfamiliar with the Matlab language will have little difficult navigating the user-friendly interface, and users with Matlab programming experience can adapt and customize MSiReader for their own needs.

A New Family of Multiferrocene Complexes with Enhanced Control of Structure and Stoichiometry via Coordination-Driven Self-Assembly and Their Electrochemistry

The design and synthesis of a new family of multiferrocene complexes with enhanced control of structure and stoichimetry via coordination-driven self-assembly is described. Insight into the structure and electronic properties of all supramolecules was obtained by electrochemical studies. The collective results provide an enhanced understanding of the influence of structural factors on the electrochemistry of multifunctional electroactive supramolecular architectures.

Mass Spectrometry-Based Biomarker Discovery: Toward a Global Proteome Index of Individuality

Biomarker discovery and proteomics have become synonymous with mass spectrometry in recent years. Although this conflation is an injustice to the many essential biomolecular techniques widely used in biomarker-discovery platforms, it underscores the power and potential of contemporary mass spectrometry. Numerous novel and powerful technologies have been developed around mass spectrometry, proteomics, and biomarker discovery over the past 20 years to globally study complex proteomes (e.g., plasma). However, very few large-scale longitudinal studies have been carried out using these platforms to establish the analytical variability relative to true biological variability. The purpose of this review is not to cover exhaustively the applications of mass spectrometry to biomarker discovery, but rather to discuss the analytical methods and strategies that have been developed for mass spectrometry-based biomarker-discovery platforms and to place them in the context of the many challenges and opportunities yet to be addressed.

Charge-state reduction with improved signal intensity of oligonucleotides in electrospray ionization mass spectrometry

The shift of charge states of oligonucleotide negative ions formed in electrospray ionization mass spectrometry to higher mass-to-charge ratio has been accomplished by addition of organic acids and bases to the solution to be electrosprayed. The use of acetic acid or formic acid combined with piperidine and imidazole effectively reduced charge states. Signal intensity and stability were enhanced greatly when the infused solution contained a high percentage of acetonitrile. In addition, the cocktail that contained imidazole, piperidine, and acetic acid in 80% acetonitrile not only reduced charge states, but also substantially suppressed Na adduction. Several oligonucleotides that varied in base composition and length were investigated, and studies of mixtures showed a significant reduction in spectral complexity.

Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis

The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (Vmax/km) for any of the complexes is 70–6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex–mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis.

A dual electrospray ionization source combined with hexapole accumulation to achieve high mass accuracy of biopolymers in fourier transform ion cyclotron resonance mass spectrometry

A dual electrospray ionization (ESI) source employed with hexapole accumulation and gated trapping provides a novel method of using an internal standard to achieve high mass accuracies in Fourier transform ion cyclotron resonance mass spectrometry. Two ESI emitters are sequentially positioned in front of the heated metal capillary inlet by a solenoid fitted to an XYZ micromanipulator; one emitter contains the analyte(s) of interest and the other an internal standard. A 5 V transistor-transistor logic pulse from the data station controls the solenoid by means of a solid-state relay so that matching of spectral peak intensities (i.e., analyte and internal standard intensities) can be accomplished by adjusting the hexapole accumulation time for each species. Polythymidine, d(pT)18, was used as the internal standard for all studies reported here. The absolute average error for an internally calibrated 15-mer oligonucleotide (theoretical monoisotopic mass = 4548.769 Da) was −1.1 ppm (external calibration: 41 ppm) with a standard deviation of ±3.0 ppm (external calibration: ±24 ppm) for a total of 25 spectra obtained at various hexapole accumulation time ratios. Linear least squares regression analysis was carried out and revealed a linear dependence of the magnitudes of the peak height ratios (analyte/internal standard) vs. hexapole accumulation time ratios (analyte/internal standard) which is described by the following equation:y = 0.45x −0.02. The fitted line had a %RSD of the slope of 28% with anR2 of 0.93. The applicability of this methodology was extended to a polymerase chain reaction product with a theoretical average molecular mass of 50,849.20 Da. With the internal standard, d(pT)18, an absolute average error of −8.9 ppm (external calibration: 44 ppm) based on five measurements was achieved with a standard deviation of 11 ppm (external calibration: ±36 ppm), thus illustrating this method’s use for characterizing large biomolecules such as those encountered in genomics and proteomics related research.

Nanoelectrospray mass spectrometry using non‐metalized, tapered (50 → 10 μm) fused‐silica capillaries

Nanoelectrospray emitter tips, pulled from fused-silica capillaries requiring no chemical treatment or metal coating, have been operated using remote coupling of the electrospray voltage. Using common laboratory implements, 50 μm i.d. fused-silica capillaries were pulled to ca. 10 μm, and attached to a stainless steel capillary via a teflon coupler for the production of nanoelectrospray (i.e. nL/min flowrates). Using a variety of different tips, flowrates were determined to range from 30 to 80 nL/min. Spectra of bovine insulin (ca. 6 kDa) and Angiotensin III (ca. 1 kDa) produced signal-to-noise (S/N) ratios of ca.500 and ca.100, respectively; each analysis consuming ca. 10 femtomoles of sample. Radical cation formation, in addition to the monoprotonated molecule was also observed during nanoelectrospray of angiotensin III which implies that this nanoelectrospray source should be applicable to trace amounts of non-polar compounds. The stability of the ion source is demonstrated by measuring the current of the electrosprayed ions at the shutter of the Fourier transform ion cyclotron resonance mass spectrometer as a function of time. Overall stability of the nanoelectrospray for angiotensin III, using no pressure to initialize or stabilize the electrospray, was determined as 23 pA ± 0.97 pA (4.2 % relative standard deviation) over a 30 minute period.

Intact and top-down characterization of biomolecules and direct analysis using infrared matrix-assisted laser desorption electrospray ionization coupled to FT-ICR mass spectrometry

We report the implementation of an infrared laser onto our previously reported matrix-assisted laser desorption electrospray ionization (MALDESI) source with ESI post-ionization yielding multiply charged peptides and proteins. Infrared (IR)-MALDESI is demonstrated for atmospheric pressure desorption and ionization of biological molecules ranging in molecular weight from 1.2 to 17 kDa. High resolving power, high mass accuracy single-acquisition Fourier transform ion cyclotron resonance (FT-ICR) mass spectra were generated from liquid- and solid-state peptide and protein samples by desorption with an infrared laser (2.94 µm) followed by ESI post-ionization. Intact and top-down analysis of equine myoglobin (17 kDa) desorbed from the solid state with ESI post-ionization demonstrates the sequencing capabilities using IR-MALDESI coupled to FT-ICR mass spectrometry. Carbohydrates and lipids were detected through direct analysis of milk and egg yolk using both UV- and IR-MALDESI with minimal sample preparation. Three of the four classes of biological macromolecules (proteins, carbohydrates, and lipids) have been ionized and detected using MALDESI with minimal sample preparation. Sequencing of O-linked glycans, cleaved from mucin using reductive β-elimination chemistry, is also demonstrated.

Coordination-driven self-assembly of cavity-cored multiple crown ether derivatives and poly[2]pseudorotaxanes

The synthesis of a new 120 degree diplatinum(II) acceptor unit and the self-assembly of a series of two-dimensional metallacyclic polypseudorotaxanes that utilize both metal-ligand and crown ether-dialkylammonium noncovalent interactions are described. Judiciously combining complementary diplatinum(II) acceptors with bispyridyl donor building blocks, with an acceptor and/or donor possessing a pendant dibenzo[24]crown-8 (DB24C8) moiety, allows for the formation of three new rhomboidal bis-DB24C8, one new hexagonal tris-DB24C8, and four new hexakis-DB24C8 metallacyclic polygons in quantitative yields. The size and shape of each assembly, as well as the location and stoichiometry of the DB24C8 macrocycle, can be precisely controlled. Each polygon is able to complex two, three, or six dibenzylammonium ions without disrupting the underlying metallacyclic polygons, thus producing eight different poly[2]pseudorotaxanes and demonstrating the utility and scope of this orthogonal self-assembly technique. The assemblies are characterized with one-dimensional multinuclear ((1)H and (31)P) and two-dimensional ((1)H-(1)H COSY and NOESY) NMR spectroscopy as well as mass spectrometry (ESI-MS). Further analysis of the size and shape of each assembly is obtained through molecular force-field simulations. (1)H NMR titration experiments are used to establish thermodynamic binding constants and poly[2]pseudorotaxane/dibenzylammonium stoichiometries. Factors influencing the efficiency of poly[2]pseudorotaxane formation are discussed.