Analytical Solution for the Electric Field Inside Dynamically Harmonized FT-ICR Cell.

Dynamically harmonized FT-ICR cell has a saddle-like hyperbolic field distribution inside when averaged over a cyclotron trajectory around the axis of the cell. Such a field distribution makes the motion along the magnetic field independent of the motion in the x,y-plane, as well as the cyclotron motion independent of the magnetron motion and prevents any disintegration of excited coherent ion clouds, which is ruining the resolution in the other types of FT-ICR cells providing by this ideal phasing of single-m/z ion clouds in the entire volume of the cell. FT-ICR instruments with such a cell show resolutions of more than ten million at m/z 1000 at relatively small magnetic fields like 7 Tesla in quadrupole detection mode, what is not reachable by any other type of modern mass spectrometers. We have found that for such ion traps, it is possible to find the analytical solution in the working volume of the trap without any averaging. The potential distribution for the almost whole volume of such a cell can be presented in the form ϕ(x, y, z) = αz2 + f2D(x, y), where f2D(x, y) is the solution of 2D Poisson equation, which could be found by the method of conformal transformation. This solution is applicable in the practical case and can serve as a base for an analytical theory of signal detection using such cells and as a standard for solutions obtained by numerical simulations of the cell field.

Tracking the Magnetron Motion in FT-ICR Mass Spectrometry.

In Fourier transform ion cyclotron resonance spectrometry (FT-ICR MS) the ion magnetron motion is not usually directly measured, yet its contribution to the performance of the FT-ICR cell is important. Its presence is manifested primarily by the appearance of even-numbered harmonics in the spectra. In this work, the relationship between the ion magnetron motion in the ICR cell and the intensities of the second harmonic signal and its sideband peak in the FT-ICR spectrum is studied. Ion motion simulations show that during a cyclotron motion excitation of ions which are offset to the cell axis, a position-dependent radial drift of the cyclotron center takes place. This radial drift can be directed outwards if the ion is initially offset towards one of the detection electrodes, or it can be directed inwards if the ion is initially offset towards one of the excitation electrodes. Consequently, a magnetron orbit diameter can increase or decrease during a resonant cyclotron excitation. A method has been developed to study this behavior of the magnetron motion by acquiring a series of FT-ICR spectra using varied post-capture delay (PCD) time intervals. PCD is the delay time after the capture of the ions in the cell before the cyclotron excitation of the ion is started. Plotting the relative intensity of the second harmonic sideband peak versus the PCD in each mass spectrum leads to an oscillating "PCD curve". The position and height of minima and maxima of this curve can be used to interpret the size and the position of the magnetron orbit. Ion motion simulations show that an off-axis magnetron orbit generates even-numbered harmonic peaks with sidebands at a distance of one magnetron frequency and multiples of it. This magnetron offset is due to a radial offset of the electric field axis versus the geometric cell axis. In this work, we also show how this offset of the radial electric field center can be corrected by applying appropriate DC correction voltages to the mantle electrodes of the ICR cell while observing the signals of the second harmonic peak group. The field correction leads to a definite performance increase in terms of resolving power and mass accuracy, and the mass spectrum contains intensity-minimized even-numbered harmonics. This is very important in the case of high performance cells, particularly the dynamically harmonized cell, since the magnetron motion can severely impair the averaging effect for dynamic harmonization and can therefore reduce the resolving power.

From Supercomputer Modeling to Highest Mass Resolution in FT-ICR.

Understanding of behavior of ion ensembles inside FT-ICR cell based on the computer simulation of ion motion gives rise to the new ideas of cell designs. The recently introduced novel FT-ICR cell based on a Penning ion trap with specially shaped excitation and detection electrodes prevents distortion of ion cyclotron motion phases (normally caused by non-ideal electric trapping fields) by averaging the trapping DC electric field during the ion motion in the ICR cell. Detection times of 5 min resulting in resolving power close to 40,000,000 have been reached for reserpine at m/z 609 at a magnetic field of only 7 Tesla. Fine structures of resolved 13Cn isotopic cluster groups could be measured for molecular masses up to 5.7 kDa (insulin) with resolving power of 4,000,000 at 7 Tesla. Based on resolved fine structure patterns atomic compositions can be directly determined using a new developed algorithm for fine structure processing. Mass spectra of proteins and multimers of proteins reaching masses up to 186 kDa (enolase tetramer) could be measured with isotopic resolution. For instance, at 7 Tesla resolving power of 800,000 was achieved for enolase dimer (96 kDa) and 500,000 for molecular masses above 100 kDa. Experimental data indicate that there is practically no limit for the resolving power of this ICR cell except by collisional damping in the ultrahigh vacuum chamber.

Absorption-mode spectra on the dynamically harmonized Fourier transform ion cyclotron resonance cell.

RATIONALE: The recently designed dynamically harmonized Fourier transform ion cyclotron resonance (FT-ICR) cell creates a more harmonized electric field for the detection of the cyclotron motion of ions and prolongs the ion transient from seconds to minutes. In order to achieve its best performance, phase correction was applied in the spectra, and new advantages of the absorption-mode were revealed. METHODS: Spectra were acquired from both simple standard and complex mixtures using either narrowband or broadband mode, and the data were processed to compare the performance of the spectra in magnitude and absorption-mode. RESULTS: The research shows that phase correction works well with data from Nikolaev's new cell, which produces the maximum improvement in resolving power (2×), and improves the match with the theoretical intensities of the isotopic peaks. In addition, the harmonic peaks can be diagnosed immediately in the absorption-mode. CONCLUSIONS: The manuscript demonstrates absorption-mode spectra from Nikolaev's ICR cell, which will be of interest to the community. The improved relative peak intensities and immediate identification of harmonic peaks will facilitate data interpretation.

Human myocardial protein pattern reveals cardiac diseases.

Proteomic profiles of myocardial tissue in two different etiologies of heart failure were investigated using high performance liquid chromatography (HPLC)/Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Right atrial appendages from 10 patients with hemodynamically significant isolated aortic valve disease and from 10 patients with isolated symptomatic coronary heart disease were collected during elective cardiac surgery. As presented in an earlier study by our group (Baykut et al., 2006), both disease forms showed clearly different pattern distribution characteristics. Interesting enough, the classification patterns could be used for correctly sorting unknown test samples in their correct categories. However, in order to fully exploit and also validate these findings there is a definite need for unambiguous identification of the differences between different etiologies at molecular level. In this study, samples representative for the aortic valve disease and coronary heart disease were prepared, tryptically digested, and analyzed using an FT-ICR MS that allowed collision-induced dissociation (CID) of selected classifier masses. By using the fragment spectra, proteins were identified by database searches. For comparison and further validation, classifier masses were also fragmented and analyzed using HPLC-/Matrix-assisted laser desorption ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) mass spectrometry. Desmin and lumican precursor were examples of proteins found in aortic samples at higher abundances than in coronary samples. Similarly, adenylate kinase isoenzyme was found in coronary samples at a higher abundance. The described methodology could also be feasible in search for specific biomarkers in plasma or serum for diagnostic purposes.

Fine structure in isotopic peak distributions measured using a dynamically harmonized Fourier transform ion cyclotron resonance cell at 7 T.

The fine structure of isotopic peak clusters in mass spectra of reserpine and substance P are measured using Fourier transform ion cyclotron resonance mass spectrometry at a 7 T magnetic field. The resolved peaks in the fine structure consist of (13)C, (15)N, (17)O, (18)O, (2)H, (33)S, (34)S, and combinations of them. A recently introduced high-resolution ion cyclotron resonance cell (Nikolaev, E. N.; Boldin, I. A.; Jertz, R.; Baykut, G. J. Am. Soc. Mass Spectrom. 2011, 22, 1125-1133) is used in these experiments. The positions of the experimentally obtained fine structure peaks on the mass scale agree with the isotopic distribution simulations with ≤200 ppb error. Some deviation from the theoretical isotopic distribution is observed, less abundant peaks in the fine structure patterns are a little suppressed compared to the larger ones. We present a method for atomic composition determination using accurate mass data and fine isotopic structure of the mass spectrum. Our method combines the traditional atomic composition determination from accurate mass data by enumeration of all possible formulas within constraints defined a priori with the estimation of element coefficients from resolved isotopic structures. These estimated values allow one to narrow the search space for the composition and therefore to reduce the number of candidate formulas.

Initial experimental characterization of a new ultra-high resolution FTICR cell with dynamic harmonization.

A new Fourier transform ion cyclotron resonance (FTICR) cell based on completely new principles of formation of the effective electric potential distribution in Penning type traps, Boldin and Nikolaev (Proceedings of the 58th ASMS Conference, 2010), Boldin and Nikolaev (Rapid Commun Mass Spectrom 25:122-126, 2011) is constructed and tested experimentally. Its operation is based on the concept of electric potential space-averaging via charged particle cyclotron motion. Such an averaging process permits an effective electric force distribution in the entire volume of a cylindrical Penning trap to be equal to its distribution in the field created by hyperbolic electrodes in an ideal Penning trap. The excitation and detection electrodes of this new cell are shaped for generating a quadratic dependence on axial coordinates of an averaged (along cyclotron motion orbit) electric potential at any radius of the cyclotron motion. These electrodes together with the trapping segments form a cylindrical surface like in a conventional cylindrical cell. In excitation mode this cell being elongated behaves almost like an open cylindrical cell of the same length. It is more effective in ion motion harmonization at larger cyclotron radii than a Gabrielse et al.-type (Int J Mass Spectrom Ion Processes 88:319-332, 1989) cylindrical cell with four compensation sections. A mass resolving power of more than twenty millions of reserpine (m/z 609) and more than one million of highly charged BSA molecular ions (m/z 1357) has been obtained in a 7T magnetic field.

Applying a dynamic method to the measurement of ion mobility.

A dynamic method is applied to measure the mobility of gas-phase ions in the dual ion funnel interface of the electrospray source of a quadrupole orthogonal time-of-flight mass spectrometer. In a new operational mode, a potential barrier was formed in the second ion funnel of the mass spectrometer and then progressively increased. In this region, a flow of gas drags the ions into the mass spectrometer while the electric force applied by the potential barrier decelerates them. Ions with lower mobility can be carried by the gas flow more easily than those with high mobility. Thus, electrical forces can block the more mobile ions more easily. Hence, the electric barrier formed in the ion funnel permits only ions below a certain mobility threshold to enter the mass spectrometer. When the barrier voltage is increased, this threshold moves from high to low mobilities. Ions with mobilities above the threshold cannot enter the mass spectrometer, and their signal decreases to zero. Thus, in a barrier voltage scan, mass spectrometric signals of ions sequentially disappear. Differentiation of these decreasing ion signal curves produces peaks from which an ion mobility spectrum can be reconstructed. Blocking voltages, i.e., the positions of the peaks on the barrier voltage scale are directly related to the mobility of these ions. An internal calibration using ions with known mobility values helps determine the unknown ion mobilities and allows calculation of ionic cross sections.

Imaging of peptides in the rat brain using MALDI-FTICR mass spectrometry.

Analytical methods are pursued to measure the identity and location of biomolecules down to the subcellular (microm) level. Available mass spectrometric imaging methods either compromise localization accuracy or identification accuracy in their analysis of surface biomolecules. In this study, imaging FTICR-MS is applied for the spatially resolved mass analysis of rat brain tissue with the aim to optimize protein identification by the high mass accuracy and online MS/MS capabilities of the technique. Mass accuracies up to 6 ppm were obtained in the direct MALDI-analysis of the tissue together with a spatial resolution of 200 microm. The spatial distributions of biomolecules differing in mass by less than 0.1 Da could be resolved, and are shown to differ significantly. Online MS/MS analysis of selected ions was demonstrated. A comparison of the FTICR-MS imaging results with stigmatic TOF imaging on the same sample is presented. To reduce the extended measuring times involved, it is recommended to restrict the FTICR-MS analyses to areas of interest as can be preselected by other, faster imaging methods.

Two-fold efficiency increase by selective excitation of ions for consecutive activation by ion-electron reactions and vibrational excitation in tandem fourier transform ion cyclotron resonance mass spectrometry.

A new technique called selective excitation of ions for consecutive activation (SEICA) is proposed for obtaining complementary fragmentation mass spectra from the same precursor ion population. SEICA utilizes precursor ions remaining intact after electron capture dissociation or another ion-electron reaction for efficient MS/MS based on a vibrational excitation (VE) technique, such as infrared multiphoton dissociation. SEICA uses the ability of ion-trapping instruments to detect product ions while retaining inside the trap intact precursor ions, making the latter available for consecutive activation by a VE technique. The possibility of practical implementation of SEICA by software-only modification of a commercial instrument is demonstrated. A 2-fold increase in the efficiency is achieved for both "single-scan" and "multiple-scan" experiments. This improvement can be particularly important for high-sensitivity applications in, for example, proteomics, where limited ECD efficiency poses an obstacle for broad implementation of this technique.

Fast gas-phase hydrogen/deuterium exchange observed for a DNA G-quadruplex.

The gas-phase hydrogen/deuterium (H/D) exchange kinetics of DNA G-quadruplexes has been investigated using Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The quadruplex [(TGGGGT)4 . 3NH4+] undergoes very fast H/D exchange, in both the positive and in the negative ion modes, compared to DNA duplexes and other quadruplexes tested, and compared to the corresponding single-stranded TGGGGT. Substitution of NH4+ for K+ did not alter this fast H/D exchange, indicating that the hydrogens of the ammonium ions are not those exchanged. However, stripping of the interior cations of the quadruplex by source collision-induced dissociation (CID) in the positive ion mode showed that the presence of the inner cations is essential for the fast exchange to be possible. Molecular dynamics simulations show that the G-quadruplex is very rigid in the gas phase with NH4+ ions inside the tetrads. We suggest that the fast H/D exchange is favored by this rigid quadruplex conformation. This example illustrates that the concept that compact DNA structures exchange H for D slower than unfolded ones is a misconception.

Coupling of fully automated chip electrospray to Fourier transform ion cyclotron resonance mass spectrometry for high-performance glycoscreening and sequencing.

The NanoMate robot has been coupled to a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer at 9.4 T and implemented for the first time for complex carbohydrate analysis. It was optimized in the negative ion mode to achieve automated sample delivery on the chip along with increased sensitivity, ultra-high resolution and accurate mass determination. A novel bracket has been designed to allow a reliable mounting of the NanoMate to the Apollo electrospray ionization (ESI) source of an APEX II instrument. The notably higher efficiency of ionization for compositional mapping of complex mixtures and feasibility for fragmentation analysis of components by sustained off-resonance irradiation collision-induced tandem mass spectrometry (SORI-CID MS2) has been demonstrated on a glycoconjugate mixture containing O-glycosylated sialylated peptides from urine of a patient suffering from a hereditary N-acetylhexosaminidase deficiency (Schindler's disease), previously analyzed by capillary-based nanoESI-FTICRMS, and of a healthy control person. Due to its potential to generate highly charged ionic species, reduce the in-source fragmentation, increase sensitivity, reproducibility and ionization efficiency, along with the ability to generate a sustained and constant electrospray, this method can be considered as a new platform for advanced glycomics.

Peptide and protein characterization by high-rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry.

The analytical utility of the electron capture dissociation (ECD) technique, developed by McLafferty and co-workers, has substantially improved peptide and protein characterization using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The limitations of the first ECD implementations on commercial instruments were eliminated by the employment of low-energy electron-injection systems based on indirectly heated dispenser cathodes. In particular, the ECD rate and reliability were greatly increased, enabling the combination of ECD/FTICR-MS with on-line liquid separation techniques. Further technique development allowed the combination of two rapid fragmentation techniques, high-rate ECD and infrared multiphoton dissociation (IRMPD), in a single experimental configuration. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation and led to improved peptide and protein characterization. The application of high-rate ECD/FTICR-MS has demonstrated its power and unique capabilities in top-down sequencing of peptides and proteins, including characterization of post-translational modifications, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation). Analysis of peptide mixtures has been accomplished using high-rate ECD in bottom-up mass spectrometry based on mixture separation by liquid chromatography and capillary electrophoresis. This paper summarizes the current impact of high-rate ECD/FTICR-MS for top-down and bottom-up mass spectrometry of peptides and proteins.

Electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry in the electron energy range 0-50 eV.

Electron capture dissociation (ECD) of polypeptide cations was obtained with pencil and hollow electron beams for both sidekick and gas-assisted dynamic ion trapping (GADT) using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with an electrostatic ion transfer line. Increasing the number of trapped ions by multiple ICR trap loads using GADT improved the ECD sensitivity in comparison with sidekick ion trapping and ECD efficiency in comparison with single ion trap load by GADT. Furthermore, enhanced sensitivity made it possible to observe ECD in a wide range of electron energies (0-50 eV). The degree, rate and fragmentation characteristics of ECD FTICR-MS were investigated as functions of electron energy, electron irradiation time, electron flux and ion trapping parameters for this broad energy range. The results obtained show that the rate of ECD is higher for more energetic (>1 eV) electrons. Long electron irradiation time with energetic electrons reduces average fragment ion mass and decreases efficiency of formation of c- and z-type ions. The obtained dependencies suggest that the average fragment ion mass and the ECD efficiency are functions of the total fluence of the electron beam (electron energy multiplied by irradiation time). The measured electron energy distributions in low-energy ECD and hot ECD regimes are about 1 eV at full width half maximum in employed experimental configurations.

Combined infrared multiphoton dissociation and electron capture dissociation with a hollow electron beam in Fourier transform ion cyclotron resonance mass spectrometry.

An electron injection system based on an indirectly heated ring-shaped dispenser cathode has been developed and installed in a 7 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This new hardware design allows high-rate electron capture dissociation (ECD) to be carried out by a hollow electron beam coaxial with the ion cyclotron resonance (ICR) trap. Infrared multiphoton dissociation (IRMPD) can also be performed with an on-axis IR-laser beam passing through a hole at the centre of the dispenser cathode. Electron and photon irradiation times of the order of 100 ms are required for efficient ECD and IRMPD, respectively. As ECD and IRMPD generate fragments of different types (mostly c, z and b, y, respectively), complementary structural information that improves the characterization of peptides and proteins by FTICR mass spectrometry can be obtained. The developed technique enables the consecutive or simultaneous use of the ECD and IRMPD methods within a single FTICR experimental sequence and on the same ensemble of trapped ions in multistage tandem (MS/MS/MS or MS(n)) mass spectrometry. Flexible changing between ECD and IRMPD should present advantages for the analysis of protein digests separated by liquid chromatography prior to FTICRMS. Furthermore, ion activation by either electron or laser irradiation prior to, as well as after, dissociation by IRMPD or ECD increases the efficiency of ion fragmentation, including the w-type fragment ion formation, and improves sequencing of peptides with multiple disulfide bridges. The developed instrumental configuration is essential for combined ECD and IRMPD on FTICR mass spectrometers with limited access into the ICR trap.

Fourier transform ion cyclotron resonance mass spectrometry with NanoLC/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis.

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.

A combined ion source for fast switching between electrospray and matrix-assisted laser desorption/ionization in Fourier transform ion cyclotron resonance mass spectrometry.

A new ion source has been developed for Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) that enables quick changes between matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) modes. When operating as an ESI source, the sample solution is sprayed through an angled nebulizer. The generated ions pass through a glass capillary followed by a skimmer and three sequential hexapole ion guides. Ions can be accumulated in the third hexapole (storage hexapole) before they are injected into the ICR trap. The second hexapole is mounted on a movable platform which also carries the MALDI sample plate. During the switch from ESI to MALDI, this platform moves the second hexapole out of the hexapole series and locates a MALDI sample plate with 384 sample positions into the area directly in front of the storage hexapole. The storage hexapole is in a medium pressure chamber (MPC) which has windows both for the incoming laser beam and for the observation optics, as well as a gas tube for pulsing collision gas into the chamber. During the MALDI operation the focused laser beam enters the MPC, passes between the hexapole rods and irradiates a MALDI sample on the target plate. The sample molecules are desorbed/ionized into the storage hexapole and simultaneously cooled by collisions with the pulsed gas. Ions desorbed from multiple laser shots can be accumulated in this hexapole before they are transferred to the ICR trap. With the combined ion source a computer-controlled switch between MALDI and ESI modes is possible in less than a minute, depending on the position of the MALDI target on the 384-spot plate. Immediate acquisition of mass spectra is possible after mode switching without the need for tuning or re-calibration.

In-source H/D exchange and ion-molecule reactions using matrix assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry with pulsed collision and reaction gases.

Controlled in-source ion-molecule reactions are performed for the first time in an external matrix assisted laser desorption ionization (MALDI) source of a Fourier transform ion cyclotron resonance mass spectrometer. The MALDI source with a hexapole ion guide that was originally designed to incorporate pulsed gas to collisionally cool ions (Baykut, G.; Jertz, R.; Witt, M. Rapid Commun. Mass Spectrom. 2000, 14, 1238-1247) has been modified to allow the study of in-source ion-molecule reactions. Upon laser desorption, a reaction gas was introduced through a second inlet and allowed to interact with the MALDI-generated ions trapped in the hexapole ion guide. Performing ion-molecule reactions in the high pressure range of the ion source prior to analysis in the ion cyclotron resonance (ICR) cell allows to maintain the ultra high vacuum in the cell which is crucial for high mass resolution measurements. In addition, due to the reaction gas pressure in the hexapole product ion formation is much faster than would be otherwise possible in the ICR cell. H/D exchange reactions with different peptides are investigated, as are proton-bound complex formations. A typical experimental sequence would be ion accumulation in the hexapole ion guide from multiple laser shots, addition of cooling gas during ion formation, addition of reaction gas, varied time delays for the ion-molecule reactions, and transmission of the product ions into the ICR cell for mass analysis. In this MALDI source H/D exchange reactions for different protonated peptides are investigated, as well as proton-bound complex formations with the reaction gas triethylamine. Amino acid sequence, structural flexibility and folding state of the peptides can be seen to play a part in the reactivity of such ions.