Scrambling of autoinducing precursor peptides investigated by infrared multiphoton dissociation with electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry

Two synthetic precursor peptides, H₂N-CVGIW and H₂N-LVMCCVGIW, involved in the quorum sensing of Lactobacillus plantarum WCFS1, were characterized by mass spectrometry (MS) with electrospray ionization and 7-T Fourier transform ion cyclotron resonance (ESI-FTICR) instrument. Cell-free bacterial supernatant solutions were analyzed by reversed-phase liquid chromatography with ESI-FTICR MS to verify the occurrence of both pentapeptide and nonapeptide in the bacterial broth. The structural characterization of both protonated peptides was performed by infrared multiphoton dissociation using a continuous CO₂ laser source at a wavelength of 10.6 μm. As their fragmentation behavior cannot be directly derived from the primary peptide structure, all anomalous fragments were interpreted as neutral loss of amino acids from the interior of both peptides, i.e., loss of V, G, VG and M, MC, V, CC, from H₂N-CVGIW and H₂N-LVMCCVGIW, respectively. Mechanisms of this scrambling are proposed. FTICR MS provides accurate masses of all fragment ions with very low absolute mass errors (<1.6 ppm), which facilitated the reliable assignment of their elemental compositions. The resolving power was more than sufficient to resolve closely isobaric product ions with routine subparts per million mass accuracies. Only the occurrence of pentapeptide was found in the cell-free culture of L. plantarum, grown in Waymouth’s medium broth, with a low content of 5.2?±?2.6 μM by external calibration. Most of it was present as oxidized H₂N-CVGIW, that is, the soluble disulfide pentapeptide with a level tenfold higher (i.e., 50?±?4 μM, n?=?3).

Fourier transform ion cyclotron resonance detection of multiphoton ionization spectroscopy

Fourier transform ion cyclotron resonance (FT-ICR) detection was tested for resonanceenhanced multiphoton ionization (REMPI) spectroscopy. The (2+1) REMPI spectra of acetaldehyde were obtained in the wavelength range 364–354 nm via a two-photon resonant 3s ← n Rydberg transition. The space-charge effects on the REMPI spectra were examined in the vicinity of the 0 ₀ ⁰ transition. The trapping efficiency measurement shows that all the ions produced from REMPI dissociation processes are arrested in the ion cyclotron resonance cell even in the presence of space-charge interactions. Axial kinetic energy release distributions of ions were extracted from the trapping efficiency data obtained under a new space-charge-free condition. FT-ICR peak heights were measured as a function of pressure at different laser powers, magnetic field strengths, and ion excitation methods to test for the detection linearity. The FT-ICR detection responds linearly to the number of ions in a low pressure limit. The product branching ratio was measured by using various ion excitation methods and was compared with the previous quadrupole mass spectrometric study. FT-ICR detection yields the mass-selected REMPI spectra and the product branching ratio in the absence of kinetic shifts.     

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.     

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.     

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.     

Endgroup determination of synthetic polymers by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) was performed on a Fourier transform ion cyclotron resonance mass spectrometer for the endgroup and monomer mass determination of three poly(oxyalkylene)s in the mass range of 400–8000 Da. A combined use of the multiple charge states observed with ESI, leads to a threefold increase in accuracy of the endgroup and monomer determination. The improvement is attributed to the increased number of datapoints used for the regression procedure, yielding more accurate results. Endgroup masses are determined with a mass error better than 5 and 75 millimass units for the molecular weight range of 400–4200 and 6200–8000 Da, respectively. A mass error of better than 1 millimass unit was observed for all monomer mass determinations. With ESI, endgroup and monomer masses have been determined for poly(ethylene glycol) oligomers with a mass higher than 8000 Da. This is almost two times higher than observed with matrix-assisted laser desorption/ ionization on the same instrument.     

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.     

Analysis of strychnos alkaloids using electrospray ionization Fourier transform ion cyclotron resonance multi-stage tandem mass spectrometry

The fragmentations of four strychnos alkaloids have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the positive ion mode. Experiments using multi-stage tandem mass spectrometry (ESI-FT-ICR-MSn) allowed us to obtain precise elemental compositions of product ions at high mass resolution. The experimental data demonstrated that the nitrogen bridge and the coordinated oxygen atom on the nitrogen bridge in the alkaloid compounds were the active sites in the MS2 fragmentations. The loss of CH3 or the OCH3 group in those alkaloids, which have an OCH3 substituent, was the dominant fragmentation mode in the MS3 fragmentations. Logical fragmentation schemes for strychnos alkaloids have been proposed and these should be useful for the identification of these compounds.     

Fragmentation study of peptides using Fourier transform ion cyclotron resonance with infrared multiphoton dissociation: experiment and simulation

In this study, the fragmentation of gas-phase protonated Angiotensin II is investigated using electrospray ionization (ESI), Fourier-transform ion cyclotron resonance (FT-ICR), and mass spectrometry (MS) with a laser cleavage infrared multiphoton dissociation (IRMPD) technique. The experimental results show that the spectra peaks for the photoproducts are y2/b6- and y7-type ions, corresponding to the cleavage of His-Pro and Asp-Arg in the parent amino acid sequence. The fragmentation of the peptide under collision-free vacuum conditions is modeled using molecular dynamics simulations (MD). The binding energy for the peptide bonds (C'-N bond) of Angiotensin II is estimated from ab initio calculations. The calculations are directed at predicting experimental measurements of the product ions from the photodissociation of the peptide. The product distributions simulated by the MD dissociation trajectories include predominantly y7/b1 and y2/b6 pair ions.     

Genotyping short tandem repeats using flow injection and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Characterizing polymerase chain reaction (PCR) amplicons has been accomplished for the first time using flow injection analysis coupled to electrospray ionization mass spectrometry (ESI-MS). The PCR amplicons were amplified at the human tyrosine hydroxylase short tandem repeat locus from an individual homozygotic for the 9.3 allele. One product was amplified using Pfu polymerase and yielded a blunt-ended amplicon of 82 base-pairs (bp) in length. The second PCR product was amplified using Taq polymerase that resulted in an amplicon with cohesive termini of 82 bp plus either mono- or diadenylation. The two PCR amplicons were alternatively injected using a 0.5-microL loop at 2 microM for the Pfu amplicon and 1 microM for the Taq amplicon with a flow rate of 200 nL/min during data acquisition. Both PCR amplicons were accurately identified using mass measurements illustrating the compatibility of ESI-MS for genotyping short tandem repeat sequences and the potential for high-throughput genotyping of large PCR amplicons.     

Direct high-resolution peptide and protein analysis by desorption electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

We report the first coupling of a desorption electrospray ionization (DESI) ion source to Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) for high-resolution protein analysis. The DESI FT-ICR-MS source design is described in detail along with preliminary data obtained on peptides and proteins ranging from 1 to 5.7 kDa.     

A novel coaxial electrospray ionization method for characterizing hexacosanoylceramides by Fourier transform ion cyclotron resonance mass spectrometry

Because of poor aqueous solubility and lack of UV chromophores, the characterization of long-chain hydrocarbons and ceramides by conventional UV and mass spectrometric methods has not been successful. Therefore, a novel coaxial electrospray ionization method was developed for characterizing reaction products of phytosphingosine and hexacosanoic acid in toluene and tetrahydrofuran (THF), by high resolving power Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). Simultaneous spraying of a solution of apolar analytes and polar reagents into the gas phase readily enabled protonation and/or sodiation of analyte with enhanced signal-to-noise (S/N). Sample introduction was by direct infusion such that the sprayers were arranged either along the instrument line-of-sight (for monospray and coaxial spray modes) or in a 45 degrees configuration for dual-spray mode. For dual-spray and coaxial spraying, p-toluenesulphonic acid was used as a reagent and sprayed simultaneously with the analyte dissolved in toluene or THF. Compounds were characterized by accurate mass measurement of the protonated and/or sodiated molecules.     

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.     

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.     

Characterization of oligomeric compounds in secondary organic aerosol using liquid chromatography coupled to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The components of secondary organic aerosols (SOAs) generated from the gas‐phase ozonolysis of two C₁₀H₁₆‐terpenes (α‐pinene; sabinene) and a cyclic C₆H₁₀ alkene (cyclohexene) were characterized by the use of a Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source operated in the negative ion mode. Reversed‐phase high‐performance liquid chromatography was used to achieve chromatographic separation of highly oxidized organic compounds. In addition to the well‐known group of low molecular weight oxidation products (monomers; e.g. dicarboxylic acids), higher molecular weight compounds (dimers) were also detected and their exact elemental compositions were determined. In order to provide additional information for the structural elucidation of these compounds, collision‐induced dissociation was applied. Based on the MS/MS spectra, two higher molecular weight products are proposed to be an ester and a peroxide. Molecular formulae calculated from the exact masses show that the SOA‐compounds are heavily oxidized and this information creates the background to a discussion of potential reaction pathways for the formation of higher molecular weight compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

High-resolution electrospray ionization fourier transform mass spectrometry with infrared multiphoton dissociation of glucokinase from Bacillus stearothermophilus

Glucokinase (GK, EC 2.7.1.2), a member of the enzyme family of hexokinases, has been shown to be linked to maturity-onset diabetes of the young type II (MODY-2). Although nucleotide and amino acid sequence information are available for the human varieties, they are not known for the variety from Bacillus stearothermophilus, which is often used in protein binding studies. Here, a combination of electrospray Fourier transform mass spectrometry (FTMS) and infrared multiphoton dissociation (IRMPD) is used to obtain accurate molecular weight and preliminary amino acid sequence information for the protein. Electrospray FTMS provides evidence of a solution phase dimer. In addition, dithiothreitol reduction shows no shift in high-resolution isotopic distributions, indicating a probable absence of disulfide bonds in the protein. The partial sequence information obtained from IRMPD could be the basis for creating a DNA probe for the protein.     

Direct characterization of intact polypeptides by matrix-assisted laser desorption electrospray ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometry

We report the characterization of a recently introduced hybrid ionization source, matrix-assisted laser desorption electrospray ionization (MALDESI), coupled to a quadrupole Fourier transform ion cyclotron resonance mass spectrometry (QFT-ICR-MS) system. We first demonstrate the ability of MALDESI-QFT-ICR MS to directly analyze and provide high mass measurement accuracy (approximately 1 part-per-million) of a polypeptide using internal calibration. Second, we show the potential of MALDESI-QFT-ICR MS for the top-down characterization of multiply charged polypeptide cations. Finally, we demonstrate sub-femtomole detection limits in MALDESI-QFT-ICR MS using a combination of naturally occurring peptides and their respective stable isotope labeled forms. The results presented herein demonstrate the feasibility of several potential applications for MALDESI-QFT-ICR MS for the direct analysis of intact biological molecules.     

Speciation of M+3-hydroxypyrone chelation complexes by electrospray ionization ion trap and Fourier transform ion cyclotron resonance mass spectrometry

Kojic acid (5-hydroxy-2-hydroxymethyl-4-pyrone) is known to have a high affinity for transition metals, and it and its derivatized cogeners are used both analytically and clinically. The interactions between kojic acid (KA) and eleven +3 metals (Al(+3), As(+3), Cr(+3), Ga(+3), Fe(+3), In(+3), Yb(+3), Y(+3), Gd(+3), Nd(+3), La(+3)) were examined by electrospray ionization mass spectrometry (ESI-MS) using an ion trap in an aqueous medium. For a subset of five ions, Fourier transform ion cyclotron resonance (FTICR)-MS was conducted to provide accurate mass confirmation of peak assignments for metals having clustering of abundant isotopes. KA readily formed complexes with all the metal ions tested. The most common complexes observed were ML(3)H(+) and M(2)L(5). Different behavior was seen for small and large ionic radius ions, with a relative cut-off between In(+3) ( approximately 80 pm) and Yb(+3) ( approximately 87 pm); a striking trend in % collision energy vs. cluster complexity was revealed. The KA-Cr(+3)complex shows a high affinity for H(2)O molecules in the gas phase, whilst In(+3) shows a preference for dimetal complexes and Y(+3) a deviant behavior when complexed to two neutrals.