Desorption/ionization of biomolecules from aqueous solutions at atmospheric pressure using an infrared laser at 3 μm

A new atmospheric pressure (AP) infrared (IR) matrix-assisted laser desorption/ionization (MALDI) ion source was developed and interfaced with a Thermo Finnigan LCQ ion trap mass spectrometer. The source utilized a miniature all-solid-state optical parametric oscillator (OPO)-based IR laser system tunable in the lambda = 1.5-4 microm spectral range and a nitrogen ultraviolet (UV) laser (lambda = 337 nm) for use in comparative studies. The system demonstrated comparable performance at 3 microm and 337 nm wavelengths if UV matrices were used. However, AP IR-MALDI using a 3 microm wavelength showed good performance with a much broader choice of matrices including glycerol and liquid water. AP IR-MALDI mass spectra of peptides in the mass range up to 2000 Da were obtained directly from aqueous solutions at atmospheric conditions for the first time. A potential use of the new AP IR-MALDI ion source includes direct MS analysis of biological cells and tissues in a normal atmospheric environment as well as on-line coupling of mass spectrometers with liquid separation techniques.     

Improved ion extraction from a linear octopole ion trap: SIMION analysis and experimental demonstration

Externally generated ions are accumulated in a linear octopole ion trap before injection into our 9.4 T Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer. Such instrumental configuration has previously been shown to provide improved sensitivity, scan rate, and duty cycle relative to accumulated trapping in the ICR cell. However, inefficient ion ejection from the octopole currently limits both detection limit and scan rate. SIMION 7.0 analysis predicts that a dc axial electric field inside the linear octopole ion trap expedites and synchronizes the efficient extraction of the octopole-accumulated ions. Further SIMION analysis optimizes the ion ejection properties of each of three electrode configurations designed to produce a near-linear axial potential gradient. More efficient extraction and transfer of accumulated ions spanning a wide m/z range promises to reduce detection limit and increase front-end sampling rate (e.g., to increase front-end resolution for separation techniques coupled with FT-ICR mass analysis). Addition of the axial field improves experimental signal-to-noise ratio by more than an order of magnitude.     

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.     

Laser desorption in transmission geometry inside a Fourier-transform ion cyclotron resonance mass spectrometer

We report here the first application of laser desorption (LD) in transmission geometry (backside irradiation of the sample through a transparent support) inside a Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR). A probe-mounted fiber optic assembly was used to simplify the implementation of this LD technique. This setup requires little or no instrument modifications, has minimum maintenance requirements, and is relatively inexpensive to build. The performance of the probe was tested by determining the molecular weight of a commercial polystyrene standard from its matrix-assisted laser desorption/ionization (MALDI) spectrum. The measured average molecular weight is comparable to that obtained for the same sample by MALDI in the conventional top-illumination arrangement (reflection geometry) and by the manufacturer of the sample by gel permeation chromatography. The average velocities measured for ions evaporated by transmission mode LD of several neat samples are about half the velocity of those obtained by using the reflection geometry. Therefore, transmission mode irradiation of the sample holds promise to desorb ions that are easier to trap in an ICR cell. An oscillating capillary nebulizer was adapted for the deposition of analytes to improve sampling reproducibility.     

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.     

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.     

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.     

Laser-induced fluorescence of trapped gas-phase molecular ions generated by internal source matrix-assisted laser desorption/ionization in a Fourier transform ion cyclotron resonance mass spectrometer

The combination of laser-induced fluorescence with mass spectrometry opens up new possibilities both for detection purposes and for structural studies of trapped biomolecular ions in the gas phase. However, this approach is experimentally very challenging, and only a handful of studies have been reported so far. In this contribution, a novel scheme for laser-induced fluorescence measurements of ions trapped inside a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer will be introduced. It is based on an open FT-ICR cell design, continuous wave axial excitation of the fluorescence, orthogonal photon collection by fiber optics, and single photon counting detection. Rhodamine 6G ions generated by an internal matrix-assisted laser desorption/ionization source were used to develop and test the set-up. Due to photobleaching processes, the excitation laser power and the observation time window have to be carefully optimized. An ion tomography method was used to align the excitation laser. Potential applications for studying the gas-phase structure of fluorescent biomolecular ions and for investigating fluorescence resonance energy transfer of donor-acceptor pairs will be presented.     

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ETD.     

A glow discharge ion source with fourier transform ion cyclotron resonance mass spectrometric detection

A glow discharge (CD) ion source has been coupled to a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer using a four-element electrostatic lens to accelerate and focus ions generated external to the instrument’s high magnetic field into its analyzer cell. Like other CD mass spectrometers, GD-FT-ICR can provide a quantitative measure of bulk analyte concentration with good precision and accuracy. Although detection limits currently attainable are several orders of magnitude higher than the commercially available magnetic sector-based instrument, CD-FT-ICR holds promise for ultrahigh resolving power elemental mass analysis. Several schemes are proposed to lower the detection limits of the technique while still providing high enough resolution to resolve isobaric interferences.     

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.     

Analysis of cancer cell lipids using matrix‐assisted laser desorption/ionization 15‐T Fourier transform ion cyclotron resonance mass spectrometry

A combination of methodologies using the extremely high mass accuracy and resolution of 15‐T Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometry (MS) was introduced for the identification of intact cancer cell phospholipids. Lipids from a malignant glioma cell line were initially analyzed at a resolution of >200 000 and identified by setting the mass tolerance to ±1 mDa using matrix‐assisted laser desorption/ionization (MALDI) 15‐T FT‐ICR MS in positive ion mode. In most cases, a database search of potential lipid candidates using the exact masses of the lipids yielded only one possible chemical composition. Extremely high mass accuracy (<0.1 ppm) was then attained by using previously identified lipids as internal standards. This, combined with an extremely high resolution (>800 000), yielded well‐resolved isotopic fine structures allowing for the identification of lipids by MALDI 15‐T FT‐ICR MS without using tandem mass spectrometric (MS/MS) analysis. Using this method, a total of 38 unique lipids were successfully identified. Copyright © 2012 John Wiley & Sons, Ltd.     

Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) fourier transform ion cyclotron resonance mass spectrometry

We report the coupling of a hybrid ionization source, matrix-assisted laser desorption electrospray ionization (MALDESI), to a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS). The details of the source design and initial data are presented. Analysis of peptides and proteins ranging from 1 to 8.6 kDa resulted in high resolving power single-acquisition FT-ICR mass spectra with average charge-states highly correlated to those obtained by nanoESI, thus, providing strong evidence that the ESI process dictates the observed charge-state distribution. Importantly, unlike the recently introduced electrospray assisted laser desorption ionization (ELDI) source reported by Shiea and coworkers [1, 2], the data we have obtained to date rely on the use of an organic acid matrix. The results presented herein provide insight into the charging mechanism of this emerging ionization approach, while also expanding the utility of FT-ICR MS for top-down protein and complex mixture analysis.     

Initial Velocity Distribution of MALDI/LDI Ions Measured by Internal MALDI Source Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry

A new method for measuring the ion velocity distribution using an internal matrix-assisted laser desorption/ionization (MALDI) source Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer is described. The method provides the possibility of studying ion velocities without any influence of electric fields in the direction of the instrument axis until the ions reach the ICR cell. It also allows to simultaneously account for and to estimate not only the velocity distribution but the angular distribution as well. The method was demonstrated using several types of compounds in laser desorption/ionization (LDI) mode. Graphical Abstract

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Experimental evaluation of a hyperbolic ion trap for fourier transform ion cyclotron resonance mass spectrometry

The Penning ion trap, consisting of hyperbolically curved electrodes arranged as an unbroken ring electrode capped by two end electrodes whose interelectrode axis lies along the direction of an applied static magnetic field, has long been used for single-ion trapping. More recently, it has been used in “parametric” mode for ion cyclotron resonance (lCR) detection of off-axis ions. In this article, we describe and test a Penning trap whose ring electrode has been cut into four equal quadrants for conventional dipolar ICR excitation (on one pair of opposed ring quadrants) and dipolar ICR detection (on the other pair). In direct comparisons to a cubic trap, the present hyperbolic trap offers somewhat improved ICR mass spectral peak shape, higher mass resolving power, and comparable frequency shift as a function of trapping voltage. Mass measurement accuracy over a wide mass range is improved twofold and mass discrimination is somewhat worse than for a cubic trap. The relative advantages of parametric, dipolar, and quadrupole modes are briefly discussed in comparison to screened and unscreened cubic traps.     

Mass spectrometry of N-linked oligosaccharides using atmospheric pressure infrared laser ionization from solution

An atmospheric pressure (AP) infrared (IR) laser ionization technique, implemented on a quadrupole ion trap mass spectrometer, was used to analyze underivatized, N-linked oligosaccharides in solution. Experiments were conducted on an atmospheric pressure infrared ionization from solution (AP-IRIS) ion source which differed from previous AP IR matrix-assisted laser desorption/ionization (MALDI) interfaces in that the ion source operated in the absence of an extraction electric field with a higher power 2.94 microm IR laser. The general term 'IRIS' is used as the mechanism of ionization differs from that of MALDI, and is yet to be fully elucidated. The AP-IRIS ion source demonstrated femtomole-level sensitivity for branched oligosaccharides. AP-IRIS showed approximately 16 times improved sensitivity for oligomannose-6 and the core-fucosylated glycan M3N2F over optimal results obtainable on a AP UV-MALDI with a 2,4,6-trihydroxyacetophenone matrix. Comparison between IR and UV cases also showed less fragmentation in the IR spectrum for a glycan with a conserved trimannosyl core, core-substituted with fucose. A mixture of complex, high-mannose and sialylated glycans resulted in positive ion mass spectra with molecular ion peaks for each sugar. Tandem mass spectrometry of the sodiated molecular ions in a mixture of glycans revealed primarily glycosidic (B, Y) cleavages. The reported results show the practical utility of AP-IRIS while the ionization mechanism is still under investigation.     

An improved ion guide for external ion injection in glow discharge&#x2014;fourier transform ion cyclotron resonance mass spectrometry

To improve the existing ion transport optics of our glow discharge (GD)-Fourier transformion cyclotron resonance (FT-ICR) mass spectrometer, we simulated several ion trajectories between the GD source region and the ICR analyzer cell. These calculations suggested that a number of simple improvements, including the use of an ion flight tube and an electrically isolated conductance limit, would increase the efficiency of ion transfer through the fringing fields of the FT-ICR superconducting magnet and into the ICR analyzer cell. Ion beam intensity was monitored as a function of the distance between the GD source and the analyzer cell before and after implementing these improvements. A twentyfold improvement in the transport efficiency, as well as a fifteenfold enhancement in detected ET-ICR signals, was observed.     

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.     

Laser desorption lamp ionization source for ion trap mass spectrometry

A two‐step laser desorption lamp ionization source coupled to an ion trap mass spectrometer (LDLI‐ITMS) has been constructed and characterized. The pulsed infrared (IR) output of an Nd:YAG laser (1064 nm) is directed to a target inside a chamber evacuated to ~15 Pa causing desorption of molecules from the target's surface. The desorbed molecules are ionized by a vacuum ultraviolet (VUV) lamp (filled with xenon, major wavelength at 148 nm). The resulting ions are stored and detected in a three‐dimensional quadrupole ion trap modified from a Finnigan Mat LCQ mass spectrometer operated at a pressure of ≥ 0.004 Pa. The limit of detection for desorbed coronene molecules is 1.5 pmol, which is about two orders of magnitude more sensitive than laser desorption laser ionization mass spectrometry using a fluorine excimer laser (157 nm) as the ionization source. The mass spectrum of four standard aromatic compounds (pyrene, coronene, rubrene and 1,4,8,11,15,18,22,25‐octabutoxy‐29H,31H‐phthalocyanine (OPC)) shows that parent ions dominate. By increasing the infrared laser power, this instrument is capable of detecting inorganic compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Construction of a hybrid quadrupole/fourier transform ion cyclotron resonance mass spectrometer for versatile MS/MS above 10 kDa

Technological advancements including an open-cylindrical Penning trap with capacitively coupled ICR cell, selective ion accumulation with a resolving quadrupole, and a voltage gradient used during ion extraction from an octopole ion trap, have individually improved dynamic range and sensitivity in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Documented here is a new instrument utilizing these technologies toward the robust detection and fragmentation of biomolecules >10 kDa. Up to 55-fold enhancement in ion population by selective ion accumulation combined with 10- to 20- fold signal-to-noise improvement by application of a DC voltage gradient to an accumulation octopole during the ion transfer event offers improved signal-to-noise (or speed) of MS/MS experiments, for proteins from Methanococcus jannaschii and Saccharomyces cerevisiae whole cell lysates. After external quadrupole filtering with a 40 m/z window, three proteins were fragmented (and identified) in parallel from the database of Methanococcus jannaschii. Electron capture dissociation (ECD) of an intact yeast protein provides extensive sequence information resulting in a high degree of localization for an N-terminal acetylation. Hybrid fragmentation, infrared multiphoton dissociation (IRMPD) followed by low energy electrons (ECD), with the electron source located laterally off the z-axis and external to the magnet bore, presents a strategy for identification of proteins by means of the sequence tag approach. Automated implementation of diverse MS(n) approaches in a Q-FTMS instrument promises to help realize "top-down" proteomics in the future.