Focus on Fourier transform ion cyclotron resonance mass spectrometry

Editorial

Focus on Fourier transform ion cyclotron resonance mass spectrometry     

Fourier transform ion cyclotron resonance study of multiply charged aggregates of small singly charged peptides formed by electrospray ionization

Aggregates of singly protonated peptides formed with a nanoelectrospray ion source have been observed in the gas phase using Fourier transform ion cyclotron resonance (FT-ICR). Employment of “soft” ion sampling conditions in the source, which were developed previously to generate water clusters of biomolecules, provides significant yields of aggregates of singly protonated GGDPG ([2GGDPG + 2H]²⁺), GGEPG ([2GGEPG + 2H]²⁺), and VEPIPY (2VEPIPY + 2H]²⁺). With peptide mixtures, heteroaggregates, e.g., [GGDPG + GGEPG + 2H]²⁺ have also been observed along with the homoaggregates. These weakly bound noncovalent complexes undergo facile exothermic dissociation into the corresponding singly protonated monomer species with normal operation of the electrospray ion source. For example, the aggregates were not observed in FT-ICR experiments utilizing a conventional electrospray ionization (ESI) or fast atom bombardment source or with a quadrupolar ion trap mass spectrometer equipped with a conventional ESI source. The formation and metastability of these aggregates are dependent on highly specific intermolecular hydrogen bonding between the monomers. The amino acid sequence (DPG) of GGDPG mimics the well-known β reverse turn of proteins and semiempirical calculations show that it provides excellent hydrogen bonding sites for a protonated N-terminus amino group. Support for this conjecture is provided by the failure to observe aggregate formation of singly protonated peptides with several larger peptides, including hexaglycine and hexaalanine.     

Fourier transform ion cyclotron resonance spectroscopy

An ion cyclotron resonance (ICR) absorption spectrum has been obtained by exciting an ICR spectral segment with a fixed-frequency electric field pulse, followed by broad-band detection, digitization of the (time-domain) transient response, and digital Fourier transformation to produce the (frequency-domain) absorption spectrum. For a given signal-to-noise ratio and resolution, the FT-ICR method generates a spectrum in a time which is two orders of magnitude shorter than that required in conventional slow-sweep ICR detection. In the present example, a signal-to-noise ratio of 8:1 and a mass resolution of about 0.005 amu for CH₄⁺ (from CH₄ at a pressure of 8 X 10^?7 torr) have been achieved, using a single data acquisition period of 25.6 msec.     

Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry.

The patterns of gene expression, post-translational modifications, protein/biomolecular interactions, and how these may be affected by changes in the environment, cannot be accurately predicted from DNA sequences. Approaches for proteome characterization are generally based upon mass spectrometric analysis of in-gel digested two dimensional polyacrylamide gel electrophoresis (2-D PAGE) separated proteins, allowing relatively rapid protein identification compared to conventional approaches. This technique, however, is constrained by the speed of the 2-D PAGE separations, the sensitivity limits intrinsic to staining necessary for protein visualization, the speed and sensitivity of subsequent mass spectrometric analyses for identification, and the limited ability for accurate quantitative measurements based on differences in spot intensity. We are presently developing alternative approaches for proteomics based upon the combination of fast capillary electrophoresis, or other suitable chromatographic separations, and the high mass accuracy and sensitivity obtainable with unique Fourier transform ion cyclotron resonance (FTICR) mass spectrometers available at our laboratory. Several approaches are presently being pursued; one based upon the analysis of intact proteins and the second upon approaches for global protein digestion and accurate peptide mass analysis. Quantitation of protein/peptide levels are based on using two or more stable-isotope labeled versions of proteomes which are combined to obtain precise quantitation of relative protein abundances. We describe the status of our efforts towards the development of a high-throughput proteomics capability and present initial results for application to several microorganisms and discuss our efforts for extending the developed capability to mammalian proteomes.     

Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance

Fourier-transform ion cyclotron resonance instrumentation is uniquely applicable to an unusual new ion chemistry, electron capture dissociation (ECD). This causes nonergodic dissociation of far larger molecules (42 kDa) than previously observed (<1 kDa), with the resulting unimolecular ion chemistry also unique because it involves radical site reactions for similarly larger ions. ECD is highly complementary to the well known energetic methods for multiply charged ion dissociation, providing much more extensive protein sequence information, including the direct identification of N- versus C-terminal fragment ions. Because ECD only excites the molecule near the cleavage site, accompanying rearrangements are minimized. Counterintuitively, cleavage of backbone covalent bonds of protein ions is favored over that of noncovalent bonds; larger (>10 kDa) ions give far more extensive ECD if they are first thermally activated. This high specificity for covalent bond cleavage also makes ECD promising for studying the secondary and tertiary structure of gaseous protein ions caused by noncovalent bonding.     

Matrix-free mass spectrometric imaging using laser desorption ionisation Fourier transform ion cyclotron resonance mass spectrometry

Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions.     

Towards analytically useful two-dimensional Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) achieves high resolution and mass accuracy, allowing the identification of the raw chemical formulae of ions in complex samples. Using ion isolation and fragmentation (MS/MS), we can obtain more structural information, but MS/MS is time- and sample-consuming because each ion must be isolated before fragmentation. In 1987, Pfändler et al. proposed an experiment for 2D FT-ICR MS in order to fragment ions without isolating them and to visualize the fragmentations of complex samples in a single 2D mass spectrum, like 2D NMR spectroscopy. Because of limitations of electronics and computers, few studies have been conducted with this technique. The improvement of modern computers and the use of digital electronics for FT-ICR hardware now make it possible to acquire 2D mass spectra over a broad mass range. The original experiments used in-cell collision-induced dissociation, which caused a loss of resolution. Gas-free fragmentation modes such as infrared multiphoton dissociation and electron capture dissociation allow one to measure high-resolution 2D mass spectra. Consequently, there is renewed interest to develop 2D FT-ICR MS into an efficient analytical method. Improvements introduced in 2D NMR spectroscopy can also be transposed to 2D FT-ICR MS. We describe the history of 2D FT-ICR MS, introduce recent improvements, and present analytical applications to map the fragmentation of peptides. Finally, we provide a glossary which defines a few keywords for the 2D FT-ICR MS field.     

Manipulating internal energy of protonated biomolecules in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The internal energy of protonated leucine enkephalin has been manipulated in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry with two newly designed pump-probe experiments. Blackbody infrared radiation was applied to pump an ion population into a well-defined internal energy distribution below the dissociation threshold. Following this pumping stage, the internal energy distribution was probed using on-resonance collisional activation to dissociate the ions. These pump-probe experiments were carried out in two different ways: (a) using on-resonance collisional activation with variable kinetic energies to dissociate the ions at a constant initial ion temperature (determining the precursor ion survival percentage as a function of kinetic energy) and (b) using on-resonance collisional activation with a constant kinetic energy to dissociate the ions at variable initial ion temperatures (to investigate the ion survival yield-initial ion temperature dependence). Using this approach, a detailed study of the effects of the initial ion temperature, the probing kinetic energy and the internal energy loss rate on the effective conversion efficiency of (laboratory-frame) kinetic energy to internal energy was conducted. This conversion efficiency was found to be dependent on the initial ion temperature. Depending on the experimental conditions the conversion efficiency (for collisions with argon) was estimated to be about 4.0 +/- 1.7%, which agrees with that obtained from a theoretical modeling. Finally, the reconstructed curves of the ion survival yield versus the mode of the (final) total internal energy distribution of the activated ion population (after pump and probe events) at different pump-probe conditions reveal the internal energy content of the activated ions.     

High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer

A novel electrospray ionization/Fourier transform ion cyclotron resonance mass spectrometer based on a 7-T superconducting magnet was developed for high-resolution accurate mass measurements of large biomolecules. Ions formed at atmospheric pressure using electrospray ionization (ESI) were transmitted (through six differential pumping stages) to the trapped ion cell maintained below 10^?9 torr. The increased pumping speed attainable with cryopumping (> 10⁵ L/s) allowed brief pressure excursions to above 10^?4 torr, with greatly enhanced trapping efficiencies and subsequent short pumpdown times, facilitating high-resolution mass measurements. A set of electromechanical shutters were also used to minimize the effect of the directed molecular beam produced by the ES1 source and were open only during ion injection. Coupled with the use of the pulsed-valve gas inlet, the trapped ion cell was generally filled to the space charge limit within 100 ms. The use of 10–25 ms ion injection times allowed mass spectra to be obtained from 4 fmol of bovine insulin (M_r 5734) and ubiquitin (M_r 8565, with resolution sufficient to easily resolve the isotopic envelopes and determine the charge states. The microheterogeneity of the glycoprotein ribonuclease B was examined, giving a measured mass of 14,898.74 Da for the most abundant peak in the isotopic envelope of the normally glycosylated protein (i.e., with five mannose and two N-acetylglucosamine residues (an error of approximately 2 ppm) and an average error of approximately 1 ppm for the higher glycosylated and various H₃PO₄ adducted forms of the protein. Time-domain signals lasting in excess of 80 s were obtained for smaller proteins, producing, for example, a mass resolution of more than 700,000 for the 4⁺ charge state (m/z 1434) of insulin.     

Theory of peak coalescence in Fourier transform ion cyclotron resonance mass spectrometry

Peak coalescence, i.e. the merging of two close peaks in a Fourier transform ion cyclotron resonance (FTICR) mass spectrum at a high number of ions, plays an important role in various FTICR experiments. In order to describe the coalescence phenomenon we would like to propose a new theory of motion for ion clouds with close mass‐to‐charge ratios, driven by a uniform magnetic field and Coulomb interactions between the clouds. We describe the motion of the ion clouds in terms of their averaged drift motion in crossed magnetic and electric fields. The ion clouds are considered to be of constant size and their motion is studied in two dimensions. The theory deals with the first‐order approximation of the equations of motion in relation to dm/m, where dm is the mass difference and m is the mass of a single ion. The analysis was done for an arbitrary inter‐cloud interaction potential, which makes it possible to analyze finite‐size ion clouds of any shape. The final analytical expression for the condition of the onset of coalescence is found for the case of uniformly charged spheres. An algorithm for finding this condition for an arbitrary interaction potential is proposed. The critical number of ions for the peak coalescence to take place is shown to depend quadratically on the magnetic field strength and to be proportional to the cyclotron radius and inversely proportional to the ion masses. Copyright © 2009 John Wiley & Sons, Ltd.     

Solvation of acylium fragment ions in electrospray ionization quadrupole ion trap and Fourier transform ion cyclotron resonance mass spectrometry

In electrospray ionization (ESI) quadrupole ion trap and Fourier transform ion cyclotron resonance mass spectrometry, certain fragment ions (e.g. acylium ions) generated either during the ion transportation process (in the source interface region) or in the ion trap are found to undergo ion--molecule reactions with ESI solvent molecules (water, acetonitrile and aliphatic alcohols) to form adduct species. These unexpected solvated fragment ions severely complicate the interpretation of mass spectrometic data. High-resolution accurate mass measurements are important in establishing the elemental compositions of these adduct species and preventing erroneous data interpretation.     

Automatic internal calibration in liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry of protein digests

Accurately measured peptide masses can be used for large-scale protein identification from bacterial whole-cell digests as an alternative to tandem mass spectrometry (MS/MS) provided mass measurement errors of a few parts-per-million (ppm) are obtained. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) routinely achieves such mass accuracy either with internal calibration or by regulating the charge in the analyzer cell. We have developed a novel and automated method for internal calibration of liquid chromatography (LC)/FTICR data from whole-cell digests using peptides in the sample identified by concurrent MS/MS together with ambient polydimethylcyclosiloxanes as internal calibrants in the mass spectra. The method reduced mass measurement error from 4.3 +/- 3.7 ppm to 0.3 +/- 2.3 ppm in an E. coli LC/FTICR dataset of 1000 MS and MS/MS spectra and is applicable to all analyses of complex protein digests by FTICRMS.     

Enhanced electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of long-chain polysaccharides

A novel strategy was developed to extend the application of electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to the analysis of long-chain polysaccharides. High molecular weight polydisperse maltodextrins (poly-alpha(1-4) glucose) and dextrans (poly-alpha(1-6) glucose) were chosen as model compounds in the present study. Increased ionization efficiency of these mixtures in the positive ion mode was achieved upon modification of their reducing end with nitrogen-containing groups. The derivatization method is based on the formation of a new C--N bond between 1,6-hexamethylenediamine (HMD) and the reducing end of the polysaccharide, which exists in solution as an equilibrium between the hemiacetal and the open-ring aldehyde form. To achieve the chemical modification of the reducing end, two synthetic pathways were developed: (i) coupling of HMD by reductive amination and (ii) oxidation of the hemiacetal to lactone, followed by ring opening by HMD to yield the maltodextrin lactonamide of 1,6-hexanediamine (HMMD). Amino-functionalized polysaccharides were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR-MS) in the positive ion mode by direct flow injection. The hexamethylenediamine (HMD) and maltodextrin lactonamide of 1,6-hexanediamine (HMMD) moieties provide increased proton affinities which dramatically improve the detection of the long-chain polysaccharides by FTICR-MS. The present approach allowed for identification of single components in mixtures with prominent heterogeneity in the degree of polymerization (DP), without the need for chromatographic separation prior to MS. The high mass accuracy was essential for the unambiguous characterization of the species observed in the analyzed mixtures. Furthermore, molecular components containing up to 42 glucose residues were detected, representing the largest polysaccharide chains analyzed so far by ESI FTICR-MS.     

Simplified application of quadrupolar excitation in Fourier transform ion cyclotron resonance mass spectrometry

A new method for application of quadrupolar excitation to the trapped ion cell of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is presented. Quadrupolar excitation is conventionally applied to the two pairs of opposed electrodes that normally perform the excitation and detection functions in the FTICR experiment. Symmetry arguments and numerically calculated isopotential contours within the trapped ion cell lead to the conclusion that quadrupolar excitation can be applied to a single pair of opposed side electrodes. Examples of effective quadrupolar axialization via this method include a sevenfold signal-to-noise enhancement derived from 50 remeasurements of a single population of trapped bovine insulin ions and the selective isolation of a single charge state of horse heart myoglobin after an initial measurement that revealed the presence of 14 charge states.     

Precision profiling and identification of human serum peptides using Fourier transform ion cyclotron resonance mass spectrometry

Many biomarker discovery studies are based on matrix-assisted laser desorption/ionisation (MALDI) peptide profiles. In this study, 96 human serum samples were analysed on a Bruker solariX(TM) MALDI Fourier transform ion cyclotron resonance (FTICR) system equipped with a 15 tesla magnet. Isotopically resolved peptides were observed in ultrahigh resolution FTICR profiles up to m/z 6500 with mass measurement errors (MMEs) of previously identified peptides at a sub-ppm level. For comparison with our previous platform for peptide profile mass analysis (i.e. Ultraflex II) the corresponding time-of-flight (TOF) spectra were obtained with isotopically resolved peptides up to m/z 3500. The FTICR and TOF systems performed rather similar with respect to the repeatability of the signal intensities. However, the mass measurement precision improved at least 10-fold in ultrahigh resolution data and thus simplified spectral alignment necessary for robust and quantitatively precise comparisons of profiles in large-scale clinical studies. From each single MALDI-FTICR spectrum an m/z-list was obtained with sub-ppm precision for all different species, which is beneficial for identification purposes and interlaboratory comparisons. Furthermore, the FTICR system allowed new peptide identifications from collision-induced dissociation (CID) spectra using direct infusion of reversed-phase (RP) C(18)-fractionated serum samples on an electrospray ionisation (ESI) source.     

Liquid chromatography and electron-capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry

Liquid separation methods in combination with electrospray mass spectrometry as well as the recently introduced fragmentation method electron capture dissociation (ECD) have become powerful tools in proteomics research. This paper presents the results of the first successful attempts to combine liquid chromatography (LC) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) with ECD in the analysis of a mixture of standard peptides and of a bovine serum albumin tryptic digest. A novel electron injection system provided conditions for ECD sufficient to yield extensive sequence information for the most abundant peptides in the mixtures on the time-scale of the chromatographic separation. The results suggest that LC/ECD-FTICRMS can be employed in the characterization of peptides in enzymatic digests of proteins or protein mixtures and identify and localize posttranslational modifications.     

Electron capture dissociation of polypeptides using a 3 Tesla Fourier transform ion cyclotron resonance mass spectrometer

Electron capture dissociation (ECD) of polypeptides has been demonstrated using a commercially available 3 Tesla Fourier transform ion cyclotron resonance (FTICR) instrument. A conventional rhenium filament, designed for high-energy electron impact ionisation, was used to effect ECD of substance P, bee venom melittin and bovine insulin, oxidised B chain. A retarding field analysis of the effective electron kinetic energy distribution entering the ICR cell suggests that one of the most important parameters governing ECD for this particular instrument is the need to employ low trapping plate voltages. This is shown to maximise the abundance of low-energy electrons. The demonstration of ECD at this relatively low magnetic field strength could offer the prospect of more routine ECD analysis for the wider research community, given the reduced cost of such magnets and (at least theoretically) the greater ease of electron/ion cloud overlap at lower field.