An external source 7 T Fourier transform ion cyclotron resonance mass spectrometer with electrostatic ion guide

An external source 7 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer offers three main novel features. First, a 9-way ion-source cross allows for mounting of up to three ionization sources simultaneously, thereby minimizing 'downtime' for changing ion sources. Second, an electrostatic (wire-in-cylinder) ion guide transports the ions approximately 1.5 m from the ion source to the ion trap for mass analysis, through a large magnetic field gradient. Third, the system operates from a modular data system described elsewhere in this issue. Luteinizing hormone-releasing hormone (LH-RH) matrix-assisted laser desorption/ionization (MALDI) FTICR positive-ion mass spectra exhibit signal-to-noise ratio greater than 1000:1 and mass resolving power, m/delta m 50% > 100,000. Laser-induced fragmentation of bradykinin demonstrates the ability of the ion guide to transmit both molecular and fragment ions simultaneously. Ultra-high resolution (average resolving power approximately 400,000) was achieved for poly(ethylene glycol) of specified number-average molecular weight, Mn approximately 3400. Future installation of an electrospray source to the ion-source cross should allow for better characterization of the performance of the ion guide.     

Enhanced mass resolving power, sensitivity, and selectivity in laser desorption Fourier transform ion cyclotron resonance mass spectrometry by ion axialization and cooling

Ion cooling and axialization produced by azimuthal quadrupolar excitation in the presence of ion-neutral collisions are applied to laser desorption Fourier transform ion cyclotron resonance mass spectrometry (LD/FT/ICR-MS). With this technique, the large kinetic and internal energies of ions generated by laser desorption processes can be cooled effectively by collisions of ions with neutral argon atoms (at > 5 x 10(-7) Torr). After sufficient cooling in the source compartment of a dual ion trap, the axialized ions may be transferred to the analyzer compartment for detection at much lower pressure (and thus much higher mass resolving power). Enhancements in both FT/ICR mass resolving power and sensitivity are observed; moreover, ion isolation with high selectivity at high pressure is also demonstrated.     

Counting individual sulfur atoms in a protein by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry: experimental resolution of isotopic fine structure in proteins

A typical molecular ion mass spectrum consists of a sum of signals from species of various possible isotopic compositions. Only the monoisotopic peak (e.g., all carbons are 12C; all nitrogens are 14N, etc.) has a unique elemental composition. Every other isotope peak at approximately integer multiples of approximately 1 Da higher in nominal mass represents a sum of contributions from isotope combinations differing by a few mDa (e.g., two 13C vs. two 15N vs. one 13C and one 15N vs. 34S, vs. 18O, etc., at approximately 2 Da higher in mass than the monoisotopic mass). At sufficiently high mass resolving power, each of these nominal-mass peaks resolves into its isotopic fine structure. Here, we report resolution of the isotopic fine structure of proteins up to 15.8 kDa (isotopic 13C,15N doubly depleted tumor suppressor protein, p16), made possible by electrospray ionization followed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass analysis at 9.4 tesla. Further, a resolving power of m/Deltam50% approximately 8,000,000 has been achieved on bovine ubiquitin (8.6 kDa). These results represent a 10-fold increase in the highest mass at which isotopic fine structure previously had been observed. Finally, because isotopic fine structure reveals elemental composition directly, it can be used to confirm or determine molecular formula. For p16, for example, we were able to determine (5.1 +/- 0.3) the correct number (five) of sulfur atoms solely from the abundance ratio of the resolved 34S peak to the monoisotopic peak.     

Electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry at 11.5 tesla: instrument design and initial results

Initial results obtained using a new electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated at a magnetic field 11.5 tesla are presented. The new instrument utilized an electrostatic ion guide between the ESI source and FTICR trap that provided up to 5% overall transmission efficiency for light ions and up to 30% efficiency for heavier biomolecules. The higher magnetic field in combination with an enlarged FTICR ion trap made it possible to substantially improve resolving power and operate in a more robust fashion for large biopolymers compared to lower field instruments. Mass resolution up to 10(6) has been achieved for intermediate size biopolymers such as bovine ubiquitin (8.6 kDa) and bovine cytochrome c (12.4 kDa) without the use of frequency drift correction methods. A mass resolution of 370,000 has been demonstrated for isotopically resolved molecular ions of bovine serum albumin (66.5 kDa). Comparative measurements were made with the same spectrometer using a lower field 3.5-tesla magnet allowing the performance gains to be more readily quantified. Further improvements in pumping capacity of the vacuum system and efficiency of ion transmission from the source are expected to lead to further substantial sensitivity gains.     

Assessing enzyme substrate specificity using combinatorial libraries and electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry

A model experiment for the 'on-line' screening of substrate libraries by enzymes using combinatorial libraries in combination with electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry has been performed. The reaction between the electrophilic substrate 1-chloro-2,4-dinitrobenzene and component of a H-gamma-Glu-Cys-Xxx-OH library, catalyzed by glutathione-S-transferase, has been monitored. It shows the feasibility of 'two-dimensional' screening of substrate libraries by ESI-FTICR mass spectrometry.     

Phase correction for collision model analysis and enhanced resolving power of fourier transform ion cyclotron resonance mass spectra

Phase correction of FT-ICR data yields an absorption spectrum that offers a gain by up to a factor of 2 in mass resolving power (at half-maximum peak height), compared to conventional magnitude-mode display. That improvement is equivalent to doubling the applied magnetic field strength, without loss in signal-to-noise (S/N) ratio, provided that the time-domain data are padded with an equal number of zeroes before FFT. Our simple, visual, user-interactive algorithm quickly corrects for zero-order and first-order variation of phase with frequency. We find that the theoretical mass resolving power enhancement for pressure-limited absorption-mode over magnitude-mode line shape depends on the collision mechanism: factor of 1.40 for hard sphere vs 3(1/2) for Langevin (ion: induced dipole). Thus, the experimental enhancement in mass resolving power (factor of 1.43 +/- 0.09) for isotopically resolved peaks in the FT-ICR mass spectra of electrosprayed bovine carbonic anhydrase (approximately 29 kDa) directly supports the hard-sphere collision model. Optimal implementation of phasing requires the following: (a) a delay between excitation and detection of less than half of one sampling interval to avoid baseline "roll" and Gibb's oscillations; (b) accurate analog-to-digital conversion; (c) a sufficiently long acquisition period to yield several data points per absorption-mode peak width at half-maximum peak height; and (d) avoidance of FT-ICR apodization functions (e.g., Hamming and Hanning) that suppress the initial time-domain data. Pulsed single-frequency excitation (duration much less than the reciprocal of the Nyquist bandwidth) can eliminate higher than first-order variation of phase with frequency. Phased FT-ICR spectra should prove especially desirable for analysis of complex mixtures, for resolving isotopic distributions in electrosprayed multiply charged macromolecules and for characterizing ion collisions (and thus ion size and shape).     

Endgroup analysis of polyethylene glycol polymers by matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry

Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) by external injection of matrix-assisted laser desorbed and ionized (MALDI) polymers offers good possibilities for characterization of low molecular weight homopolymers (MW range up to 10 kDa). The molecular masses of the molecular weight distribution (MWD) components of underivatized and derivatized (dimethyl, dipropyl, dibutyl and diacetyl) polyethylene glycol (PEG) 1000 and 4000 were measured by MALDI-FTICR-MS. These measurements have been performed using a commercial FTICR spectrometer with a home-built external ion source. MALDI of the samples with a 2,5-dihydroxybenzoic acid matrix in a 1000:1 matrix-to-analyte molar ratio produces sodiated molecules in a sufficient yield to trap the ions in the ICR cell. The masses of the molecular weight distribution of PEG components were measured in broad-band mode with a mass accuracy of < 5 ppm in the mass range around 1000 u and within 40 ppm accuracy around 4000 u. From these measurements, the endgroup mass of the polymer was determined by correlation of the measured component mass with the degree of polymerization. The masses of the PEG endgroups have been determined within a deviation of 3-10 millimass units for the PEG1000 derivatives and 10-100 millimass units for the PEG4000 derivatives, thus confirming the identity of the distal parts of the model compounds.     

Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Protein tyrosine phosphatases (PTPase) play important roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. Here, solvent accessibility is determined for backbone amide protons from various segments of wild-type Yersinia PTPase in the presence or absence of 220 microM vanadate, a competitive inhibitor, as well as an active site mutant in which the essential cysteine 403 has been replaced by serine (C403S). The method consists of solution-phase H/D exchange, followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform ion cyclotron resonance mass spectrometry. Proteolytic segments spanning approximately 93.5% of the primary sequence are analyzed. Binding of vanadate reduces the H/D exchange rate throughout the protein, both for the WpD loop and for numerous other residues that are shielded when that loop is pulled down over the active site on binding of the inhibitor. The single active site C403S mutation reduces solvent access to the WpD loop itself, but opens up the structure in several other segments. Although the 3D structure of the ligand-bound C403S mutant is similar to that of the wild-type PTPase, and the C403S mutant and the wild-type enzyme display similar affinities for vanadate, the thermodynamics for binding of vanadate is different for the two proteins. Collectively, these results establish the flexibility of the WpD loop (previously inferred by comparing PTPase X-ray single-cyrstal diffraction structures in the presence and absence of a tungstate inhibitor), as well as several other signficant changes in segment exposure and/or flexibility that are not evident from X-ray structures.     

Characterization of combinatorial peptide libraries by electrospray ionization Fourier transform mass spectrometry

The advantages of Fourier transform mass spectrometry (FTMS) are precision high mass accuracy measurements and the capability of high resolution, multistage mass spectrometry together with a number of other advanced features. These powerful facilities can be used to rapidly screen complex mixtures without the necessity of chromatographic separations. The example shown here illustrates the use of the high resolving power and accurate mass capabilities of FTMS for the rapid, direct analysis of a complex mixture, which had been ionized by direct infusion electrospray ionization.     

Differentiation of brewing yeast strains by pyrolysis mass spectrometry and Fourier transform infrared spectroscopy

Two rapid spectroscopic approaches for whole-organism fingerprinting--pyrolysis mass spectrometry (PyMS) and Fourier transform infrared spectroscopy (FT-IR)--were used to analyse 22 production brewery Saccharomyces cerevisiae strains. Multivariate discriminant analysis of the spectral data was then performed to observe relationships between the 22 isolates. Upon visual inspection of the cluster analyses, similar differentiation of the strains was observed for both approaches. Moreover, these phenetic classifications were found to be very similar to those previously obtained using genotypic studies of the same brewing yeasts. Both spectroscopic techniques are rapid (typically 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of the successful discrimination of both ale and lager yeasts. We believe that these whole-organism fingerprinting methods could find application in brewery quality control laboratories.     

Synthesis of charged phenyl radicals and biradicals by laser photolysis in a Fourier-transform ion cyclotron resonance mass spectrometer

The feasibility of generating substituted phenyl radicals and biradicals (with a charged substituent) in the gas phase by laser photolysis was examined by using a Fourier-transform ion cyclotron resonance mass spectrometer. The precursors were generated by ipso-substitution of a halogen atom in the radical cation of a di- or trihalobenzene by various nucleophiles. Photolytic cleavage of the remaining carbon-halogen bond(s) with 266-nm radiation was found to produce many substituted phenyl radicals in greater yields than the earlier employed method, sustained off-resonance irradiated collision-activated dissociation (SORI-CAD). Furthermore, ion generation by photolysis leads to isomerization less often than collisional activation. Finally, not only phenyl-bromine and phenyl-iodine but also certain phenyl-chlorine bonds can be cleaved by photolysis, whereas the synthetic utility of SORI-CAD appears to be largely limited to the cleavage of phenyl-iodine bonds. Hence, laser photolysis greatly expands the variety of substituted phenyl radicals and biradicals that can be synthesized inside a mass spectrometer.     

Alkaline degradation of oligosaccharides coupled with matrix-assisted laser desorption/ionization Fourier transform mass spectrometry: a method for sequencing oligosaccharides

A new technique for determining sequence and linkage information of underivatized oligosaccharides is developed using alkaline degradation and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). Alkaline degradation (also known as the "peeling" reaction) is a chemical degradation technique that only cleaves the glycosidic bond at the reducing end by beta-elimination to yield a new reducing end. The reaction products are sampled directly with minimal cleanup and monitored by MALDI-FTMS to elucidate the oligosaccharide sequence. Linkage information is provided by cross-ring cleavage fragmentation of the new reducing ends, created by either MALDI source fragmentation or sustained off-resonance irradiation collision-induced dissociation. This method is illustrated by the successful sequence and linkage determination of neutral, branched, fucosylated, and sialylated oligosaccharides. Experiments on differently linked disaccharides are also performed to determine the specificity of the cross-ring cleavage reactions. The power of this technique is enhanced by the Fourier transform mass analyzer, which provides high-resolution, exact mass, and facile tandem mass spectrometry experiments of MALDI-produced ions.     

High-resolution electrospray ionization Fourier transform mass spectrometry with infrared multiphoton dissociation of glucokinase from Bacillus Stearothermophilus

Glucokinase (GK, EC 2.7.1.2), a member of the enzyme family of hexokinases, has been shown to be linked to maturity-onset diabetes of the young type II (MODY-2). Although nucleotide and amino acid sequence information are available for the human varieties, they are not known for the variety from Bacillus stearothermophilus, which is often used in protein binding studies. Here, a combination of electrospray Fourier transform mass spectrometry (FTMS) and infrared multiphoton dissociation (IRMPD) is used to obtain accurate molecular weight and preliminary amino acid sequence information for the protein. Electrospray FTMS provides evidence of a solution phase dimer. In addition, dithiothreitol reduction shows no shift in high-resolution isotopic distributions, indicating a probable absence of disulfide bonds in the protein. The partial sequence information obtained from IRMPD could be the basis for creating a DNA probe for the protein.     

Applications of 1.06-micron IR laser desorption on a Fourier transform mass spectrometer

Various sugars, peptides, and lipids were analyzed on a Fourier transform mass spectrometer using laser desorption and ionization with and without the assistance of matrixes. A compact Nd:YAG laser with an output at 1.06 microns corresponding to fundamental frequency was employed. Gram-negative and Gram-positive bacteria were also subjected to laser desorption mass spectrometry. Characteristics ions of conjugated lipid, formed by attachment of alkali metal cations, endogenous to the cells, were observed. Particle/liquid matrixes (e.g., cobalt in glycerol) proved to be useful with the 1.06-micron laser. The particles absorb efficiently laser radiation in a broad wavelength range. The liquid provides the same advantages as in fast atom bombardment: increased signal-to-noise ratios and enhanced sample lifetimes. The effect of laser power on total ion current was shown to differ for samples with and without the particle/liquid matrix. The Fourier transform analyzer provides MS/MS capability for both positive and negative ions from complex mixtures. Ions desorbed externally are introduced into the cell via a quadrupole ion guide with a lower mass cutoff. Such a setup allows matrix ions to be excluded and thus provides excellent signal-to-noise ratios for lower mass range fragment ions formed inside the cell.