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.     

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.     

Site-specific amide hydrogen exchange in melittin probed by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

Electron capture dissociation (ECD) has been proposed to be a non-ergodic process, i.e. to provide backbone dissociation of gas-phase peptides faster than randomization of the imparted energy. One potential consequence could be that ECD can fragment deuterated peptides without causing hydrogen scrambling and thereby provide amino acid residue-specific amide hydrogen exchange rates. Such a feature would improve the resolution of approaches involving solution-phase amide hydrogen exchange combined with mass spectrometry for protein structural characterization. Here, we explore this hypothesis using melittin, a haemolytic polypeptide from bee venom, as our model system. Exchange rates in methanol calculated from consecutive c-type ion pairs show some correlation with previous NMR data: the amide hydrogens of leucine 13 and alanine 15, located at the unstructured kink surrounding proline 14 in the melittin structure adopted in methanol, appear as fast exchangers and the amide hydrogens of leucine 16 and lysine 23, buried within the helical regions of melittin, appear as slow exchangers. However, calculations based on c-type ions for other amide hydrogens do not correlate well with NMR data, and evidence for deuterium scrambling in ECD was obtained from z*-type ions.     

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.     

Evaluation and optimization of electron capture dissociation efficiency in fourier transform ion cyclotron resonance mass spectrometry

Electron capture dissociation (ECD) efficiency has typically been lower than for other dissociation techniques. Here we characterize experimental factors that limit ECD and seek to improve its efficiency. Efficiency of precursor to product ion conversion was measured for a range of peptide (∼15% efficiency) and protein (∼33% efficiency) ions of differing sizes and charge states. Conversion of precursor ions to products depends on electron irradiation period and maximizes at ∼5–30 ms. The optimal irradiation period scales inversely with charge state. We demonstrate that reflection of electrons through the ICR cell is more efficient and robust than a single pass, because electrons can cool to the optimal energy for capture, which allows for a wide range of initial electron energy. Further, efficient ECD with reflected electrons requires only a short (∼500 μs) irradiation period followed by an appropriate delay for cooling and interaction. Reflection of the electron beam results in electrons trapped in or near the ICR cell and thus requires a brief (∼50 μs) purge for successful mass spectral acquisition. Further electron irradiation of refractory precursor ions did not result in further dissociation. Possibly the ion cloud and electron beam are misaligned radially, or the electron beam diameter may be smaller than that of the ion cloud such that remaining precursor ions do not overlap with the electron beam. Several ion manipulation techniques and use of a large, movable dispenser cathode reduce the possibility that misalignment of the ion and electron beams limits ECD efficiency.     

Electron capture dissociation implementation progress in fourier transform ion cyclotron resonance mass spectrometry

Successful electron capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) applications to peptide and protein structural analysis have been enabled by constant progress in implementation of improved electron injection techniques. The rate of ECD product ion formation has been increased to match the liquid chromatography and capillary electrophoresis timescales, and ECD has been combined with infrared multiphoton dissociation in a single experimental configuration to provide simultaneous irradiation, fast switching between the two techniques, and good spatial overlap between ion, photon, and electron beams. Here we begin by describing advantages and disadvantages of the various existing electron injection techniques for ECD in FT-ICR MS. We next compare multiple-pass and single-pass ECD to provide better understanding of ECD efficiency at low and high negative cathode potentials. We introduce compressed hollow electron beam injection to optimize the overlap of ion, photon, and electron beams in the ICR ion trap. Finally, to overcome significant outgassing during operation of a powerful thermal cathode, we introduce nonthermal electron emitter-based electron injection. We describe the first results obtained with cold cathode ECD, and demonstrate a general way to obtain low-energy electrons in FT-ICR MS by use of multiple-pass ECD.     

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.     

Focus on Fourier transform ion cyclotron resonance mass spectrometry

Editorial

Focus on Fourier transform ion cyclotron resonance mass spectrometry     

Simplified electron ejection in Fourier transform ion cyclotron resonance mass spectrometry by suspended trapping

Suspended trapping is used to eject electrons in negative-ion Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometric experiments. In contrast to electron ejection by resonant excitation of the trapping motion, suspended trapping involves allowing the electrons to escape along the z-axis (perpendicular to the trap plates) while the trapping potential is briefly removed. The duration of this event is sufficiently short (～10 μs) so that ion losses are negligible; the overall effect is that of a ‘high-pass mass filter’. Suspended trapping is simpler to implement and more generally applicable to various cell geometries than resonant electron ejection. The effectiveness of the suspended trapping technique is not compromised by the anharmonicity of the potential well in ‘elongated’ ICR traps, but depends simply on the time it takes the electrons to escape the cell. Finally, a small, positive offset potential (～+0.25 V) applied to the trap plates during the suspended trapping event increases the efficiency of the ejection.     

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.     

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.     

Electron capture dissociation of substance P using a commercially available Fourier transform ion cyclotron resonance mass spectrometer

Electron capture dissociation of the peptide Substance P is reported for the first time, with an unmodified, commercially available Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The fragmentation pattern is compared with that obtained with collisionally induced dissociation of the ions in the electrospray ion source, and note that electron capture dissociation gives a more easily interpreted spectrum, showing mainly C-fragments. With the exception of the proline residues, which require cleavage of two chemical bonds, we observe all C-fragmental we find the bias voltage of the electron gun not to be very critical.     

Fourier transform ion cyclotron resonance mass spectrometry in a high homogeneity 25 tesla resistive magnet

Many performance parameters of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry improve dramatically with increasing magnetic field. Our prior results from a 20 tesla resistive magnet showed that performance was limited by the large spatial inhomogeneity in spite of the high field. In this paper, we compare matrix-assisted laser desorption/ionization (MALDI) mass spectra at the same magnetic field for two resistive magnets with different field spatial homogeneity. In addition, we report MALDI spectra at 25 tesla—the highest magnetic field for FT-ICR to date. The first broadband FT-ICR mass spectrum [poly(ethylene glycol) 2000] from a resistive magnet is accurately fitted by the standard ICR mass calibration function.     

An antibiotic linked to peptides and proteins is released by electron capture dissociation fourier transform ion cyclotron resonance mass spectrometry

Desfuroylceftiofur (DFC) is a bioactive beta-lactam antibiotic metabolite that has a free thiol group. Previous experiments have shown release of DFC from plasma extracts after addition of a disulfide reducing agent, suggesting that DFC may be bound to plasma and tissue proteins through disulfide bonds. We have reacted DFC with [Arg(8)]-vasopressin (which has one disulfide bond) and bovine insulin (which has three disulfide bonds) and analyzed the reaction products by use of electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS), which has previously shown preferential cleavage of disulfide bonds. We observe cleavage of DFC from vasopressin and insulin during ECD, suggesting that DFC is indeed bound to peptides and proteins through disulfide bonds. Specifically, we observed dissociative loss of one, as well as two, DFC species during ECD of [vasopressin + 2(DFC-H) + 2H](2+) from a single electron capture event. Loss of two DFCs could arise from either consecutive or simultaneous loss, but in any case implies a gas phase disulfide exchange step. ECD of [insulin + DFC + 4H](4+) shows preferential dissociative loss of DFC. Combined with HPLC, ECD FT-ICR-MS may be an efficient screening method for detection of drug-biomolecule binding.     

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.     

Combined infrared multiphoton dissociation and electron-capture dissociation using co-linear and overlapping beams in Fourier transform ion cyclotron resonance mass spectrometry

A novel set-up for Fourier transform ion cyclotron resonance mass spectrometry (FTICR) is reported for simultaneous infrared multiphoton dissociation (IRMPD) and electron-capture dissociation (ECD). An unmodified electron gun ensures complete, on-axis overlap between the electron and the photon beams. The instrumentation, design and implementation of this novel approach are described. In this configuration the IR beam is directed into the ICR cell using a pneumatically actuated mirror inserted into the ion-optical path. Concept validation was made using different combinations of IRMPD and ECD irradiation events on two standard peptides. The ability to perform efficient IRMPD, ECD and especially simultaneous IRMPD and ECD using lower irradiation times is demonstrated. The increase in primary sequence coverage, with the combined IRMPD and ECD set-up, also increases the confidence in peptide and protein assignments.     

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.     

High-frequency fourier transform ion cyclotron resonance mass spectrometry

The experimental Fourier transform ion cyclotron resonance (FT/ICR) frequency range has been extended to 107 MHz. We report the observation of FT/ICR signals from electron-ionized species of mass-to-charge ratio 8, 7, 6, 5, 4, 3, 2, and 1 μ per elementary charge. We show that moderately high charge states of atomic ions (e.g., N³⁺) are easily generated and detected. Several applications for high-frequency FT/ICR mass spectrometry are proposed and discussed.     

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.