High-performance Mass Spectrometer Research Articles

Preparation and Use of an Integrated Microcapillary HPLC Column and ESI Device for Proteomic Analysis

INTRODUCTIONImplementation of separation techniques in miniaturized formats on-line with high-performance mass spectrometers and the development of miniaturized sprayers as electrospray ionization (ESI) ion sources have reduced the amount of peptide required for complete and routine sequence characterization from several picomoles to a few femtomoles and below. Arguably, much of this gain in sensitivity is due to the combination of concentration-dependent type ionization devices such as ESI and on-line capillary separation devices of very small internal diameter (I.D.). This can be primarily attributed to a reduced mass flow rate of solvents and other background constituents into the ESI source, which allows for greater sample ionization efficiency. For reasons of robustness, most microcapillary HPLC (μLC) work is done with 75- or 100-μm I.D. capillary columns that clog less frequently than 50-μm I.D. capillary columns. The following protocol describes the preparation of an integrated C(18)-packed capillary column-ESI microspray device. In this protocol, a polyimide capillary tip tapered to ~5 μm is used to hold the C(18)-derivatized particles in place.

Infrared spectroscopy of organometallic ions in the gas phase: From model to real world complexes

Gas phase mid-infrared spectroscopy of molecular ions can nowadays be performed with high performance mass spectrometers coupled to free electron lasers (FEL). The wide and continuous tunability of highly intense FELs in the mid-infrared region can be exploited for performing infrared multiple photon dissociation (IRMPD) spectroscopy of molecular ions. This review will focus on gas phase IRMPD spectroscopic investigations aiming at probing the structure and the reactivity of transition metal complexes. The performance of infrared spectroscopy for characterizing the coordination mode of polydentate ligands and the spin state of the metal will be illustrated. Infrared spectroscopy has also been exploited to probe the reactivity of metal complexes, and a special attention will be given to the infrared spectroscopy of reactive intermediates.

Relative Quantification of Stable Isotope Labeled Peptides Using a Linear Ion Trap-Orbitrap Hybrid Mass Spectrometer

The quantitative analysis of complex biological samples has emerged as a key research area in the field of proteomics. Although quantitative proteomic experiments remain challenging, these strategies have been greatly facilitated by the development of newer high-performance mass spectrometers. In this work, we have evaluated the use of the LTQ-Orbitrap, a hybrid mass spectrometer in which a linear ion trap is coupled to an Orbitrap mass analyzer, for quantitative analyses. By analyzing a range of yeast protein standards, we found that the high mass accuracy, high resolution, large ion capacity, and large dynamic range of the LTQ-Orbitrap led to as much as a 4-5-fold improvement in the number and quality of the peptide ratio measurements compared to similar analyses done on the LTQ. We also successfully quantified protein expression differences that occur in metabolically labeled rat synapses during brain development to further demonstrate the suitability of the LTQ-Orbitrap for the comparative analysis of complex tissue samples.

Comparison of a Salmonella typhimurium proteome defined by shotgun proteomics directly on an LTQ-FT and by proteome pre-fractionation on an LCQ-DUO

Shotgun proteomics is rapidly becoming one of the most efficient and popular tools to examine protein expression in cells. Numerous laboratories now have a wide array of low- and high-performance mass spectrometry instrumentation necessary to complete proteome-wide projects. Often these laboratories have time and financial constraints that prohibit all projects from being conducted on high-performance state-of-the-art mass spectrometers. Here, we compare shotgun proteomic results using a direct 'lyse, digest and analyse' approach on a high-performance mass spectrometer (i.e. the LTQ-FT) with the results from a much lower-performance instrument (i.e. the LCQ-DUO) where, for the latter, various traditional protein pre-fractionation steps and gas-phase fractionation were used to increase the proteome coverage. Our results demonstrate that shotgun proteomic analyses conducted on the lower-performance LCQ-DUO mass spectrometer could adequately characterize a PhoP constitutive strain of Salmonella typhimurium if proteome pre-fractionation steps and gas-phase fractionation were included.

Delineation of Positron Emission Tomography Imaging Agent Binding Sites on β-Amyloid Peptide Fibrils

A range of imaging agents for use in the positron emission tomography of Alzheimer's disease is currently under development. Each of the main compound classes, derived from thioflavin T (PIB), Congo Red (BSB), and aminonaphthalene (FDDNP) are believed to bind to mutually exclusive sites on the beta-amyloid (Abeta) peptide fibrils. We recently reported the presence of three classes of binding sites (BS1, BS2, BS3) on the Abeta fibrils for thioflavin T derivatives and now extend these findings to demonstrate that these sites are also able to accommodate ligands from the other chemotype classes. The results from competition assays using [3H]Me-BTA-1 (BS3 probe) indicated that both PIB and FDDNP were able to displace the radioligand with Ki values of 25 and 42 nM, respectively. BSB was unable to displace the radioligand tracer from the Abeta fibrils. In contrast, each of the compounds examined were able to displace thioflavin T (BS1 probe) from the Abeta fibrils when evaluated in a fluorescence competition assay with Ki values for PIB, FDDNP, and BSB of 1865, 335, and 600 nM, respectively. Finally, the Kd values for FDDNP and BSB binding to Abeta fibrils were directly determined by monitoring the increases in the ligand intrinsic fluorescence, which were 290 and 104 nM, respectively. The results from these assays indicate that (i) the three classes of thioflavin T binding sites are able to accommodate a wide range of chemotype structures, (ii) BSB binds to two sites on the Abeta fibrils, one of which is BS2, and the other is distinct from the thioflavin T derivative binding sites, and (iii) there is no independent binding site on the fibrils for FDDNP, and the ligand binds to both the BS1 and BS3 sites with significantly lower affinities than previously reported.

Ion mobility mass spectrometry of proteins in a modified commercial mass spectrometer

Ion mobility has emerged as an important technique for determining biopolymer conformations in solvent free environments. These experiments have been nearly exclusively performed on home built systems. In this paper we describe modifications to a commercial high performance mass spectrometer, the Waters UK “Ultima” Q-Tof, that allows high sensitivity measurement of peptide and protein cross sections. Arrival time distributions are obtained for a series of peptides (bradykinin, LHRH, substance P, bombesin) and proteins (bovine and equine cytochrome c, myoglobin, α-lactalbumin) with good agreement found with literature cross sections where available. In complex ATD’s, mass spectra can be obtained for each feature confirming assignments. The increased sensitivity of the commercial instrument is retained along with the convenience of the data system, crucial features for analysis of protein misfolding systems.

A two-dimensional quadrupole ion trap mass spectrometer

The use of a linear or two-dimensional (2-D) quadrupole ion trap as a high performance mass spectrometer is demonstrated. Mass analysis is performed by ejecting ions out a slot in one of the rods using the mass selective instability mode of operation. Resonance ejection and excitation are utilized to enhance mass analysis and to allow isolation and activation of ions for MS n capability. Improved trapping efficiency and increased ion capacity are observed relative to a three-dimensional (3-D) ion trap with similar mass range. Mass resolution comparable to 3-D traps is readily achieved, including high resolution at slower scan rates, although adequate mechanical tolerance of the trap structure is a requirement. Additional advantages of 2-D over 3-D ion traps are also discussed and demonstrated.

Use of stable isotopes to study fatty acid and lipoprotein metabolism in man

Tracer studies have long been an important tool for lipid metabolism research. Recent advances and availability of high performance mass spectrometers (MS) and improved stable isotopically labeled tracers contribute to an increase in stable isotope tracer studies in humans. We briefly review recent studies and discuss advances in high sensitivity methods and applications. GC/MS analysis. Tracer studies with gas chromatography/mass spectrometry (GC/MS) usually rely on D labeling, where labeling with more that three D atoms shifts the analyte mass above that of the natural abundance envelope, and the MS monitors selected masses representing the isotopimers of interest. Recent examples are the work of Emken and coworkers, who investigated the desaturation of 18:0 and 16:0, and 18:2n-6 and 18:3n-3 elongation/desaturation, in adults, with oral doses of about 3 g of d 2,4,6 fatty acids. They showed modest levels of 18:0 and 16:0 desaturation over 2 days and an influence of dietary 18:2n-6 on elongation. In premature infants, Salem, Uauy and coworkers recently have used d 5-18:2n-6 and d 5-18:3n-3 doses of 50–100 mg/kg body weight to show that infants as small as 1980 g and 32 weeks gestation elongate and desaturate both precursors within 24 h. Most fatty acid metabolites including 20:4n-6, 20:5n-3, and 22:6n-3 were easily detected in serum. High precision isotope ratio MS (IRMS). In 1992, we introduced a high sensitivity fatty acid tracer method based on [U- 13C] tracers and GC-combustion-IRMS (GCC-IRMS). The combustion interface facilitates carbon-by-carbon tracer detection; with U- 13C tracers all GC peaks are detected with highest precision. Rhee et al have quantified the desaturation of 18:0 and 16:0 in lipoproteins of adults using 30 mg oral doses (0.5 mg/kg). In the first 12 h, conversion of 18:0 to 18:1 was 7% in chylomicrons and 17% in VLDL, showing that both intestine and liver desaturate 18:0. Plasma conversion of 18:0 over 144 h was 14%, while that for 16:0 was 2%, showing that combined intestine and liver desaturation is minor compared with normal fluctuations in dietary levels. Carnielli applied GCC-IRMS to 8:0 elongation in very-low-birth-weight infants. Significant conversion products of 8:0 were 14:0 (5%), 16:0 (8%) but not 10:0 or 12:0.

A high-performance mass spectrometer for very small size

A small bench-top mass spectrometer of the double-focusing type is designed and constructed. The sector magnet of the instrument is very compact due to the small deflection angle (27.8°) and its weight is only less than 30 kg though the magnet radius is 15 cm and the magnetic field is 1.52 T. The field arrangement of QQQHQCQ (Q: Quadrupole lens, H: Homogeneous magnetic sector, C: Cylindrical electric sector) is adopted to achieve high performance. A mass resolution of 5000 can be obtained with an entrance slit of 84 μm.

磁場型高性能質量分析計のイオン光学

Ion optical investigation on high performance mass spectrometer is described. The field arrangement QQQOHQCQ (Q: Quadrupole, O: Octapole, H: Homogeneous magnetic sector, C: Cylindrical electric sector) is suitable for the design of mass spectrometers with ultra high resolution (AF=926,000) and with very large mass dispersion (C/AM=4). A zoom mass spectrometer is proposed by adjusting only the applied electric potentials to three quadrupoles without changing geometrical configuration. The image magnification can be changed from 0.046 to 0.187 continuously keeping small image aberrations. Very small size and high performance mass spectrometer is available by using double sector magnet. The correction of image aberrations by hexapole is discussed.

高性能二重収束質量分析計のイオン光学

Ion optics for the high performance mass spectrometer is investigated. In order to estimate the characteristics of a mass spectrometer numerically, a performance factor, PF, and an aberration factor, AF, are introduced and defined. The PF is defined as a product of four factors related to C/X (C, mass dispersion; X, image magnification), deflection angle of magnetic sector, total path length and ion beam transmission through magnet gap. Each factor consists of a ratio compared to the value for 180° magnetic analyzer. The AF is defined as a maximum resolving power calculated from the simulated image under the incident ion beam conditions of 1% spread of α, δ, y, β.Almost perfect higher order focusing and high performance are attained by mass spectrometers with the field arrangement of QQHQC and QQQHQC (Q, quadrupole; H, homogeneous magnetic sector; C, cylindrical electric sector). In these mass spectrometers, the value of PF reaches 100 and that of AF 260,000.

High-performance mass spectrometers of third-order focusing

Ion optics of high-performance mass spectrometers of double-focusing type is investigated. The characteristics of mass spectrometers are normally determined by the resolving power and the transmission of the apparatus. In addition to these, its geometrical size, cost and weight are also important factors. In order to estimate these characteristics numerically, a performance factor, PF, and an aberration factor, AF, are introduced and defined. Ion-optical systems with the field arrangement QQQOHQC (quadrupole, quadrupole, quadrupole, octupole, homogeneous magnetic sector, quadrupole, cylindrical electric sector) are investigated with the objective to obtain large PF and AF simultaneously. Almost perfect second- and third-order focusing and high performance are attained.

High performance mass spectrometers of magnetic sector type

The ion optics of high performance mass spectrometers of double focusing type are investigated. In order to estimate the characteristics of a mass spectrometer numerically, a performance factor, PF, and an aberration factor, AF, are introduced and defined, and ion optical systems with larger PF and AF are sought. Excellent focusing and very high performance are attained by mass spectrometers with the field arrangement quadrupole, quadrupole, octapole, homogeneous magnetic sector, quadrupole, cylindrical electric sector (QQOHQC). Several examples are shown.

Critical appearance size of doubly charged Xe clusters revisited

In previous experiments on doubly charged Xe clusters, no Xe2+n cluster ions were observed for n less than 53 atoms and no Xe3+n for n less than 114 atoms. From recent computer simulations it was inferred that Xe2+51 ions have lifetimes ∼100 ps while Xe2+55 ions have lifetimes ∼10 μs in agreement with these previous experimental results. Using a high performance mass spectrometer we have repeated these experiments. In contrast to these earlier findings, we are here able to observe doubly charged Xe cluster ions with sizes down to n2=47 (at lifetimes of ∼400 μs). The critical appearance size for triply charged Xe clusters is n3=108. In accordance with a recently suggested sequential ionization mechanism for the production of stable doubly charged van der Waals cluster ions, the appearance energy of Xe2+n was found to lie well below the appearance energy of Xe2+ from Xe.

Improvement of mass spectrometers

Recent progress in the improvement of mass spectrometers at Osaka University is presented. Mass spectrometers are designed by using the computer program TRIO, which calculates the ion trajectories in the third order approximation. Several high-performance mass spectrometers are proposed and some of them have been constructed and examined. Two different kinds are described in detail: one is a single focusing mass spectrometer with large incident and exit angles and the other is a double focusing mass spectrometer with field arrangement QQHQC (quadrupole, quadrupole, homogeneous magnetic sector, quadrupole, cylindrical electric sector). High resolution and high sensitivity are attained simultaneously.

Fundamental aspects and applications of fourier-transform ion-cyclotron resonance spectrometry

The operating principles, features, advantages, applications and potential of Foourier-transform ion-cyclotron resonance (F.t./i.c.r. or F.t.m.s.) mass spectrometry are discussed. It is shown that F.t./i.c.r. technology creates a high-performance mass spectrometer with high speed, high sensitivity, ultrahigh mass resolution, very wide mass range and unparalleled versatility.

Computer-Aided Design of a New High-Performance Mass Spectrometer

A new procedure for designing a high-performance mass spectrometer is described. TRIO-FIT, a computer program which utilizes an automatic fitting function, is employed for the purpose. As an example of TRIO-FIT's usefulness, an ion-optical design for a high-performance mass spectrometer (high resolution, mass range, and transmission) is developed from several alternatives. A mass resolution of 300,000 and a mass range of up to M/Z 200,000 can be expected from this system. A method of examining the practicability of a given mass spectrometer design is also described, and this procedure is demonstrated on the proposed design of the high performance instrument.

Quality of mass and intensity measurements from a high performance mass spectrometer

Quality of mass and intensity measurements from a high performance mass spectrometer