Consecutive infrared multiphoton dissociations in a fourier transform ion cyclotron resonance mass spectrometer

Consecutive infrared multiphoton dissociations (IRMPD) may be observed in a Fourier transform ion cyclotron resonance mass spectrometer (FTICR). This is the IRMPD equivalent of previous MS(n)() experiments using CID. This work presents a versatile technique, using a bistable shutter to gate ON and OFF a continuous-wave (CW) CO(2) laser for multiple irradiation periods of 0.1-1000 s duration. Consecutive photodissociations, up to MS(4), are demonstrated for the proton-bound dimer of diethyl ether and the resulting fragment ions. The photoproducts are formed close to the center of the FTICR cell, resulting in high product ion recovery efficiency. This differs from CID products, which are formed throughout the FTICR cell causing reisolation/detection problems. The fragmentation resulting from the use of low-intensity, CW, infrared laser radiation is shown to be much more energy selective than CID. Photodissociation of C(2)H(5)OH(2)(+) ion produces the lowest energy product ion exclusively, even though the two product channels differ only by ～5 kcal/mol. Low-energy CID, however, produces a mixture of C(2)H(5)(+) and H(3)O(+) products in the ratio of 1.3:1. Hence, the higher energy pathway (C(2)H(5)(+)) is substantially favored. The current results indicate that this IRMPD MS(n)() technique may be successfully applied to large biomolecules prepared by electrospray or MALDI.     

Characterization of laser-induced acoustic desorption coupled with a Fourier transform ion cyclotron resonance mass spectrometer

Several experimental factors have been investigated that influence the efficiency of desorption and subsequent chemical ionization of nonvolatile, thermally labile molecules during laser-induced acoustic desorption/Fourier transform ion cyclotron resonance mass spectrometry (LIAD/FT-ICR) experiments. The experiments were performed by using two specially designed LIAD probes of different outer diameters (1/2 and 7/8 in.) and designs. Several improvements to the design of the "first generation" (1/2 in.) LIAD probe are presented. The larger diameter (7/8 in.) probe provides a larger surface area for desorption than the smaller diameter probe. Further, it was designed to desorb molecules on-axis with the magnetic field of the instrument. This is in contrast to the smaller probe for which desorption occurs 1.3 mm off-axis. This improved alignment, which provides better overlap between the desorbed molecules and trapped reagent ions, results in a substantial increase in the sensitivity of LIAD analyses. The thickness of the sample layer deposited on the irradiated metal foil and the number of laser shots fired on the backside of the foil were found to have a significant effect on the overall signal and the relative abundances of the ions formed in the experiment. Evaporation of a tetrapeptide, Val-Ala-Ala-Phe (VAAF), from Ag, Al, Au, Cu, Fe, and Ti foils, followed by protonation by protonated pyridine, revealed that the titanium foil provides the greatest signal. The importance of the laser power density was examined by desorbing a low MW polymer, polyisobutenyl succinic anhydride, at power densities ranging from 5.40 x 10(8) to 9.00 x 10(8) W/cm(2) at the backside of the foil. Higher laser power densities resulted in greater signals and an improved distribution for the higher molecular weight oligomers.     

Laser microprobe with fourier transform ion cyclotron resonance mass spectrometer for surface analysis

Fourier transform ion cyclotron resonance laser microprobe mass spectrometry (FTICR LMMS) uses focused laser irradiation of solids with a spot of 5 microm and a FTICR mass analyzer for local analysis with high mass resolution. A new ion source design has been developed to improve the extraction and transfer of ions generated in an external laser microprobe source. Calculations predicted trapping of ions initially emitted with angles up to 40 degrees and 60 degrees from the surface and from a distance of 1 mm above the sample, respectively. The analytical performances of the method have been verified on two sets of test samples. First, detection of chemisorbed benzotriazole on copper, average of two monolayers, has been shown with less sample consumption than typically required in static secondary ion mass spectrometry with a time-of-flight analyzer. Second, experiments on a thermal plate for offset printing have shown the feasibility of analysis and quantification of dyes embedded in a polymer matrix.     

Epitope mapping of a 95 kDa antigen in complex with antibody by solution-phase amide backbone hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry

The epitopes of a homohexameric food allergen protein, cashew Ana o 2, identified by two monoclonal antibodies, 2B5 and 1F5, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) and the results were compared to previous mapping by immunological and mutational analyses. Antibody 2B5 defines a conformational epitope, and 1F5 defines a linear epitope. Intact murine IgG antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen-monoclonal antibody (Ag-mAb) complexes. mAb-complexed and uncomplexed (free) rAna o 2 were then subjected to HDX. HDX instrumentation and automation were optimized to achieve high sequence coverage by protease XIII digestion. The regions protected from H/D exchange upon antibody binding overlap and thus confirm the previously identified epitope-bearing segments: the first extension of HDX monitored by mass spectrometry to a full-length antigen-antibody complex in solution.     

Interfacing a polymer-based micromachined device to a nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometer

Here we report the design, fabrication, and operation of a polymer-based microchip device interfaced to a nanoelectrospray ionization source and a Fourier transform ion cyclotron resonance mass spectrometer. The poly(methyl methacrylate) micromachined device was fabricated using X-ray lithography to produce a network of channels with high aspect ratios. Fabrication of high aspect ratio channels allows for zero dead volume interfaces between the microchip platform and the nanoelectrospray capillary interface. The performance of this device was evaluated with standard peptide and protein samples. High-quality mass spectral data from peptide and proteins (and mixtures thereof) were obtained without any interfering chemical noise from the polymer or the developers and plasticizers used in the fabrication process. Sample cross-contamination is not a problem using this polymer-based microchip device as demonstrated by the sequential analysis of several proteins. The nanoelectrospray source was operated at flow rates from 20 to 100 nL/min using pressure-driven flow, and uninterrupted operation for several hours is demonstrated without any noticeable signal degradation. The ability to fabricate multiple devices using injection molding or hot-embossing techniques of polymers provides a lower cost alternative to silica-based devices currently utilized with mass spectrometry.     

Surface-induced dissociation in a Fourier transform ion cyclotron resonance mass spectrometer: instrument design and evaluation

A new Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) has been constructed in our laboratory. The instrument employs surface-induced dissociation (SID) as an activation method for obtaining structural information on biomolecules in the gas phase. Tandem SID mass spectra can be acquired using either a continuous or a pulsed mode of operation. Collision energy of precursor ion is controlled by a dc offset of the ICR cell. This approach eliminates defocusing of the ion beam by the ion-transfer optics as a function of ion kinetic energy and constitutes a significant improvement over our previous experimental setup. Furthermore, it can be easily implemented on any FTICR mass spectrometer. Very high signal-to-noise ratios of 200-500 were obtained in single-scan SID mass spectra of model peptides with acquisition time less than 1.1 s. Reasonable SID signal was detected in single-scan spectra with total acquisition time of only 0.3 s. The high signal-to-noise ratio and the fast acquisition time point on a potential application of SID for high-throughput studies in FTICR MS.     

Surface-induced dissociation of ions produced by matrix-assisted laser desorption/ionization in a fourier transform ion cyclotron resonance mass spectrometer

Intermediate pressure matrix-assisted laser desorption/ionization (MALDI) source was constructed and interfaced with a 6-T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially configured for surface-induced dissociation (SID) studies. First MALDI-SID results in FT-ICR are presented, demonstrating unique advantages of SID over conventional FT-ICR MS ion activation techniques for structural characterization of singly protonated peptide ions. Specifically, we demonstrate that SID on a diamond surface results in a significantly better sequence coverage for singly protonated peptides than SORI-CID. A combination of two effects contributes to the improved sequence coverage: shattering of peptide ions on surfaces opens up a variety of dissociation channels at collision energies above 40 eV, and second, wide internal energy distribution deposited by collision with a stiff diamond surface provides an efficient mixing between the primary reaction channels that are dominant at low internal energies and extensive fragmentation at high internal excitation that results from shattering. Activation of MALDI-generated ions by collisions with surfaces in FT-ICR MS is a new powerful method for characterization and identification of biomolecules     

Design and characterization of a high-power laser-induced acoustic desorption probe coupled with a fourier transform ion cyclotron resonance mass spectrometer

We report here the construction and characterization of a high-power laser-induced acoustic desorption (LIAD) probe designed for Fourier transform ion cyclotron resonance mass spectrometers to facilitate analysis of nonvolatile, thermally labile compounds. This "next generation" LIAD probe offers significant improvements in sensitivity and desorption efficiency for analytes with larger molecular weights via the use of higher laser irradiances. Unlike the previous probes which utilized a power-limiting optical fiber to transmit the laser pulses through the probe, this probe employs a set of mirrors and a focusing lens. At the end of the probe, the energy from the laser pulses propagates through a thin metal foil as an acoustic wave, resulting in desorption of neutral molecules from the opposite side of the foil. Following desorption, the molecules can be ionized by electron impact or chemical ionization. Almost an order of magnitude greater power density (up to 5.0x10(9) W/cm2) is achievable on the backside of the foil with the high-power LIAD probe compared to the earlier LIAD probes (maximum power density approximately 9.0x10(8) W/cm2). The use of higher laser irradiances is demonstrated not to cause fragmentation of the analyte. The use of higher laser irradiances increases sensitivity since it results in the evaporation of a greater number of molecules per laser pulse. Measurement of the average velocities of LIAD-evaporated molecules demonstrates that higher laser irradiances do not correlate with higher velocities of the gaseous analyte molecules.     

Preparation and in situ characterization of surfaces using soft landing in a Fourier transform ion cyclotron resonance mass spectrometer

Mass-selected peptide ions produced by electrospray ionization were deposited onto fluorinated self-assembled monolayer surfaces (FSAM) surfaces by soft landing using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially designed for studying interactions of large ions with surfaces. Analysis of the modified surface was performed in situ by combining 2-keV Cs+ secondary ion mass spectrometry with FT-ICR detection of the sputtered ions (FT-ICR-SIMS). Regardless of the initial charge state of the precursor ion, the SIMS mass spectra included singly protonated peptide ion, peptide fragment ions, and peaks characteristic of the surface in all cases. In some experiments, multiply protonated peptide ions and [M + Au]+ ions were also observed upon SIMS analysis of modified surfaces. For comparison with the in situ analysis of the modified surfaces, ex situ analysis of some of the modified surfaces was performed by 25-keV Ga+ time-of-flight-secondary ion mass spectrometry (TOF-SIMS). The ex situ analysis demonstrated that a significant number of soft-landed peptide ions remain charged on the surface even when exposed to air for several hours after deposition. Charge retention of soft-landed ions dramatically increases the ion yields obtained during SIMS analysis and enables very sensitive detection of deposited material at less than 1% of monolayer coverage. Accumulation of charged species on the surface undergoes saturation due to coulomb repulsion between charges at close to 30% coverage. We estimated that close to 1 ng of peptide could be deposited on the spot area of 4 mm2 of the FSAM surface without reaching saturation.     

Combined electron capture and infrared multiphoton dissociation for multistage MS/MS in a Fourier transform ion cyclotron resonance mass spectrometer

We have mounted a permanent on-axis dispenser cathode electron source inside the magnet bore of a 9.4-T Fourier transform ion cyclotron resonance mass spectrometer. This configuration allows electron capture dissociation (ECD) to be performed reliably on a millisecond time scale. We have also implemented an off-axis laser geometry that enables simultaneous access to ECD and infrared multiphoton dissociation (IRMPD). Optimum performance of both fragmentation techniques is maintained. The analytical utility of performing either ECD or IRMPD on a given precursor ion population is demonstrated by structural characterization of several posttranslationally modified peptides: IRMPD of phosphorylated peptides results in few backbone (b- and y-type) cleavages, and product ion spectra are dominated by neutral loss of H3PO4. In contrast, ECD provides significantly more backbone (c- and z*-type) cleavages without loss of H3PO4. For N-glycosylated tryptic peptides, IRMPD causes extensive cleavage of the glycosidic bonds, providing structural information about the glycans. ECD cleaves all backbone bonds (except the N-terminal side of proline) in a 3-kDa glycopeptide with no saccharide loss. However, only a charge-reduced radical species and some side chain losses are observed following ECD of a 5-kDa glycopeptide from the same protein. An MS3 experiment involving IR laser irradiation of the charge-reduced species formed by electron capture results in extensive dissociation into c- and z-type fragment ions. Mass-selective external ion accumulation is essential for the structural characterization of these low-abundance (modified) peptides.     

Electrically compensated Fourier transform ion cyclotron resonance cell for complex mixture mass analysis

Complex natural organic mixtures such as petroleum require ultrahigh mass spectral resolution to separate and identify thousands of elemental compositions. Here, we incorporate a custom-built, voltage-compensated ICR cell for Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS), based on a prior design by Tolmachev to produce optimal mass resolution. The compensated ICR cell installed in a custom-built 9.4 T FTICR mass spectrometer consists of seven cylindrical segments with axial proportions designed to generate a dc trapping potential that approaches an ideal three-dimensional axial quadrupolar potential. However, the empirically optimized compensation voltages do not correspond to the most quadrupolar trapping field. The compensation electrodes minimize variation in the reduced cyclotron frequency by balancing imperfections in the magnetic and electric field. The optimized voltages applied to compensation electrodes preserve ion cloud coherence for longer transient duration by approximately a factor of 2, enabling separation and identification of isobaric species (compounds with the same nominal mass but different exact mass) common in petroleum, such as C(3) vs SH(4) (separated by 3.4 mDa) and SH(3)(13)C vs (12)C(4) (separated by 1.1 mDa). The improved performance of the ICR cell provides more symmetric peak shape and better mass measurement accuracy. A positive ion atmospheric pressure photoionization (APPI) petroleum spectrum yields more than 26,000 assigned peaks, Fourier-limited resolving power of 800,000 at m/z 500 (6.6 s transient duration), and 124 part per billion root mean square (rms) error. The tunability of the compensation electrodes is critical for optimal performance.     

An automated matrix-assisted laser desorption/ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometer for "bottom-up" proteomics

Here we describe a new quadrupole Fourier transform ion cyclotron resonance hybrid mass spectrometer equipped with an intermediate-pressure MALDI ion source and demonstrate its suitability for "bottom-up" proteomics. The integration of a high-speed MALDI sample stage, a quadrupole analyzer, and a FT-ICR mass spectrometer together with a novel software user interface allows this instrument to perform high-throughput proteomics experiments. A set of linearly encoded stages allows sub-second positioning of any location on a microtiter-sized target with up to 1536 samples with micrometer precision in the source focus of the ion optics. Such precise control enables internal calibration for high mass accuracy MS and MS/MS spectra using separate calibrant and analyte regions on the target plate, avoiding ion suppression effects that would result from the spiking of calibrants into the sample. An elongated open cylindrical analyzer cell with trap plates allows trapping of ions from 1000 to 5000 m/z without notable mass discrimination. The instrument is highly sensitive, detecting less than 50 amol of angiotensin II and neurotensin in a microLC MALDI MS run under standard experimental conditions. The automated tandem MS of a reversed-phase separated bovine serum albumin digest demonstrated a successful identification for 27 peptides covering 45% of the sequence. An automated tandem MS experiment of a reversed-phase separated yeast cytosolic protein digest resulted in 226 identified peptides corresponding to 111 different proteins from 799 MS/MS attempts. The benefits of accurate mass measurements for data validation for such experiments are discussed.     

Supercritical fluid chromatography reduction of hydrogen/deuterium back exchange in solution-phase hydrogen/deuterium exchange with mass spectrometric analysis

The single biggest problem with solution-phase H/D exchange as a mass spectrometric probe of surface exposure in a protein (or protein complex) is back-exchange of H for D after the initial H/D exchange has been quenched. Back-exchange results in loss of pertinent data and also greatly hampers data analysis. Previously, very fast, cold (0-4 degrees C) HPLC was performed to help reduce back-exchange, but calculated back-exchange still averages approximately 30%. In this report, supercritical fluid chromatography replaces HPLC as the desalting/separation technique prior to mass analysis, providing a dramatic reduction in back-exchange compared to the fast, cold HPLC methods.     

Zeptomole-sensitivity electrospray ionization--Fourier transform ion cyclotron resonance mass spectrometry of proteins

Methods are being developed for ultrasensitive protein characterization based upon electrospray ionization (ESI) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The sensitivity of a FTICR mass spectrometer equipped with an ESI source depends on the overall ion transmission, which combines the probability of ionization, transmission efficiency, and ion trapping in the FTICR cell. Our developments implemented in a 3.5 tesla FTICR mass spectrometer include introduction and optimization of a newly designed electrodynamic ion funnel in the ESI interface, improving the ion beam characteristics in a quadrupole-electrostatic ion guide interface, and modification of the electrostatic ion guide. These developments provide a detection limit of approximately 30 zmol (approximately 18,000 molecules) for proteins with molecular weights ranging from 8 to 20 kDa.     

Parts-per-billion mass measurement accuracy achieved through the combination of multiple linear regression and automatic gain control in a Fourier transform ion cyclotron resonance mass spectrometer

Fourier transform ion cyclotron resonance mass spectrometry has the ability to achieve unprecedented mass measurement accuracy (MMA); MMA is one of the most significant attributes of mass spectrometric measurements as it affords extraordinary molecular specificity. However, due to space-charge effects, the achievable MMA significantly depends on the total number of ions trapped in the ion cyclotron resonance (ICR) cell for a particular measurement. Even through the use of automatic gain control (AGC), the total ion population is not constant between spectra. Multiple linear regression calibration in conjunction with AGC is utilized in these experiments to formally account for the differences in total ion population in the ICR cell between the external calibration spectra and experimental spectra. This ability allows for the extension of dynamic range of the instrument and for the mean MMA values to remain less than 1 part-per-million (ppm). In addition, multiple linear regression calibration is used to account for both differences in total ion population in the ICR cell as well as relative ion abundance of a given species, which also affords mean MMA values at the parts-per-billion (ppb) level.     

Experimental investigations of the internal energy of molecules evaporated via laser-induced acoustic desorption into a Fourier transform ion cyclotron resonance mass spectrometer

The internal energy of neutral gas-phase organic and biomolecules, evaporated by means of laser-induced acoustic desorption (LIAD) into a Fourier transform ion cyclotron resonance mass spectrometer, was investigated through several experimental approaches. The desorbed molecules were demonstrated not to undergo degradation during the desorption process by collecting LIAD-evaporated molecules and subjecting them to analysis by electrospray ionization/quadrupole ion trap mass spectrometry. Previously established gas-phase basicity values were remeasured for LIAD-evaporated organic molecules and biomolecules with the use of the bracketing method. No endothermic reactions were observed. The remeasured basicity values are in close agreement with the values reported in the literature. The amount of internal energy deposited during LIAD is concluded to be less than a few kilocalories per mole. Chemical ionization with a series of proton-transfer reagents was employed to obtain a breakdown curve for a protonated dipeptide, Val-Pro, evaporated by LIAD. Comparison of this breakdown curve with a previously published analogous curve obtained by using substrate-assisted laser desorption (SALD) to evaporate the peptide suggests that the molecules evaporated via LIAD have a similar internal energy as those evaporated via SALD.     

High-resolution field desorption/ionization fourier transform ion cyclotron resonance mass analysis of nonpolar molecules

We report the first field desorption ionization broadband high-resolution (m/Deltam(50%) approximately 65 000) mass spectra. We have interfaced a field ionization/field desorption source to a home-built 9.4-T FT-ICR mass spectrometer. The instrumental configuration employs convenient sample introduction (in-source liquid injection) and external ion accumulation. We demonstrate the utility of this configuration by generating high-resolution positive-ion mass spectra of C(60) and a midboiling crude oil distillate. The latter contains species not accessible by common soft-ionization methods, for example, low-voltage electron ionization, electrospray ionization, and matrix-assisted laser desorption/ionization. The present work demonstrates significant advantages of FI/FD FT-ICR MS for analysis of nonpolar molecules in complex mixtures.     

Parts-per-billion fourier transform ion cyclotron resonance mass measurement accuracy with a "walking" calibration equation

Ion cyclotron resonance frequency, f, is conventionally converted to ion mass-to-charge ratio, m/z (mass "calibration") by fitting experimental data spanning the entire detected m/z range to the relation, m/z = A/f + B/f(2), to yield rms mass error as low as ～200 ppb for ～10?000 resolved components of a petroleum crude oil. Analysis of residual error versus m/z and peak abundance reveals that systematic errors limit mass accuracy and thus the confidence in elemental composition assignments. Here, we present a calibration procedure in which the spectrum is divided into dozens of adjoining segments, and a separate calibration is applied to each, thereby eliminating systematic error with respect to m/z. Further, incorporation of a third term in the calibration equation that is proportional to the magnitude of each detected peak minimizes systematic error with respect to ion abundance. Finally, absorption-mode data analysis increases mass measurement accuracy only after minimization of systematic errors. We are able to increase the number of assigned peaks by as much as 25%, while reducing the rms mass error by as much as 3-fold, for significantly improved confidence in elemental composition assignment.     

Pulsed-gas glow discharge for ultrahigh mass resolution measurements with fourier transform ion cyclotron resonance mass spectrometry

A new pulsed-gas glow discharge (GD) source has been developed for use with an external ion source Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. With pulsed argon gas introduction into the GD source, the gas load and pressure in the mass analyzer region were greatly reduced; this resulted in improved mass resolution. Mass resolution of greater than 1?450?000 (fwhm) has been achieved for Cu(+) ions from a brass sample, the highest reported for any type of GD mass spectrometer. The pulsed-gas GD source promises analytical usefulness for ultrahigh resolution measurements in GD mass spectrometry.     

Identification of microcystin toxins from a strain of Microcystis aeruginosa by liquid chromatography introduction into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer

The cyclic heptapeptide microcystin toxins produced by a strain of Microcystis aeruginosa that has not been investigated previously were separated by liquid chromatography and identified by high-accuracy m/z measurements of their [M + H]+ ions and the fragment ions produced by collision-activated dissociation of the [M + H]+ ions. The cyanobacteria B2666 strain was cultured in a standard growth medium, and the toxins were released from the cells, extracted from the aqueous phase, and concentrated using standard procedures. The microcystins were separated by reversed-phase microbore liquid chromatography and introduced directly into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer with electrospray ionization. The known microcystins (MC) MC-LR, MC-LA, [MeSer7]MC-LR, MC-LL, MC-LF, and MC-L(Aba) were identified along with the two previously unreported structural variants [Asp3]MC-LA and [Asp3]MC-LL. In addition to the [M + H]+ ions, accurate m/z measurements were made of 12-18 product ions for each identified microcystin. The mean difference between measured and calculated exact m/z was less than 2 parts per million, which often allowed assignment of unique compositions to the observed ions. A mechanism is presented that accounts for an important collision-activated dissociation process that gives valuable sequence ions from microcystins that do not contain arginine. The analytical technique used in this work is capable of supporting fairly rapid and very reliable identifications of known microcystins when standards are not available and of most structural variants independent of additional information from other analytical techniques.