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.     

Improvement of electron capture efficiency by resonant excitation

A novel pulse sequence improving the efficiency for electron capture dissociation (ECD) of an unmodified Fourier transform ion cyclotron resonance (FTICR) mass spectrometer by more than an order of magnitude is presented. Commercially available FTICR instruments are usually equipped with a filament-based electron source producing an electron beam that has a rather small cross section. An ideal overlap between the rotating ion cloud and the electron beam appears to be a prerequisite for a high ECD efficiency. A reduced interception of the ion cloud and the electron beam is probably due to the contribution of the magnetron motion to the trajectory of the ions, resulting in a precession about the z-axis of the instrument. By increasing the kinetic energy and therefore increasing the cyclotron radii of the precursor ions by resonant excitation, the overlap of the rotating ion cloud with the electron beam is improved. By use of this protocol the efficiency of electron capture is substantially increased and consequently the acquisition time of ECD spectra is reduced significantly. The capability of resonant excitation of the precursor ions during the irradiation with electrons is demonstrated for standard peptides. This approach is particularly valuable for analysis and characterization of O-glycosylated peptides. In addition to amino acid sequence information, the attachment site of the labile glycan moiety is determined, and also radical-site-induced fragmentations of the glycosidic bonds are observed.     

Identification and localization of the fatty acid modification in ghrelin by electron capture dissociation

Electron capture dissociation (ECD) has been demonstrated to be an effective fragmentation technique for characterizing the site and structure of the fatty acid modification in ghrelin, a 28-residue growth-hormone-releasing peptide that has an unusual ester-linked n-octanoyl (C8:0) modification at Ser-3. ECD cleaves 21 of 23 possible backbone amine bonds, with the product ions (c and z· ions) covering a greater amino acid sequence than those obtained by collisionally activated dissociation (CAD). Consistent with the ECD nonergodic mechanism, the ester-linked octanoyl group is retained on all backbone cleavage product ions, allowing for direct localization of this labile modification. In addition, ECD also induces the ester bond cleavage to cause the loss of octanoic acid from the ghrelin molecular ion; the elimination process is initiated by the capture of an electron at the protonated ester group, which is followed by the radical-site-initiated reaction known as -cleavage. The chemical composition of the attached fatty acid can be directly obtained from the accurate Fourier transform ion cyclotron resonance (FTICR) mass measurement of the ester bond cleavage product ions.     

Characterization of phosphate-containing metabolites by calcium adduction and electron capture dissociation

Several phosphate-containing metabolites, including nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), adenosine 5'-diphosphate ribose (ADP-r), adenosine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP), have been characterized with electron capture dissociation (ECD) and sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) tandem mass spectrometry (MS/MS) in positive-ion mode. Calcium complexation was used to successfully produce abundant doubly charged cationic precursor ions with or without hydration. This approach enabled application of ECD to acidic metabolites for the first time. Fragmentation pathways observed in ECD and SORI-CAD of calcium-adducted phosphate-containing metabolites were complementary. Unique fragmentation was observed in ECD compared to SORI-CAD MS/MS, including ribose cross-ring cleavage for NAD and NADP, and generation of hydrated product ions, including cross-ring fragments, for hydrated ATP and GTP. A combination of ECD and CAD appears promising for maximizing structural information about metabolites.     

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.     

Characterization of DESI-FTICR mass spectrometry - from ECD to accurate mass tissue analysis

Implementation of desorption electrospray ionization (DESI) technique on a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is described. Desorption electrospray technique is capable of the direct investigation of natural samples without any need for sample preparation or chromatographic separation. Since the DESI mass spectra of natural samples are very complex owing to the lack of preseparation or cleanup, the ideal mass spectrometric analyzer for these applications is a high-resolution instrument such as FTICR mass spectrometer. DESI was implemented by constructing an electronically controlled source framework comprising six linear moving stages and one rotating stage. A three-dimensional linear stage was used to accommodate samples, while another 3D linear stage equipped with rotating stage was used as a spray mount. A modified electrosonic sprayer was used as a primary electrospray device. DESI-FTICR setup was characterized with regard to geometrical, electrical and flow conditions using deposited peptide samples in range of 1-100 pmol gross deposited amount on glass and polymer surfaces. Optimized conditions enabled the routine acquisition of DESI-MS spectra on the instrument at 130 000 resolution in the broadband mode and with comparable sensitivity to data reported in the literature. Since the main significance of DESI-FTICR MS is the combination of intact tissue analysis, the capabilities of the technique were demonstrated by analyzing murine liver samples. Presence of lysophospholipids in the liver tissue was tentatively associated with the lipid metabolism taking place in liver. DESI-FTICR is also a promising technique in the field of peptide analysis due to capability of top-down sequencing using electron capture dissociation. As a proof-of-principle experiment, a small synthetic polypeptide containing 36 amino acids was ionized using DESI and was sequenced in the FTICR by means of ECD (electron capture dissociation) fragmentation. Spectra gave almost full sequence information in agreement with the known amino acid sequence of the species.     

Collisionally activated dissociation and electron capture dissociation provide complementary structural information for branched permethylated oligosaccharides

Doubly charged sodiated and permethylated linear malto-oligosaccharides ({Glc}6-{Glc}9), branched N-linked glycans (high-mannose type GlcNAc2Man5-9, and complex asialo- and disialylated-biantennary glycans) were analyzed by tandem mass spectrometry using collisionally-activated dissociation (CAD) and "hot" electron capture dissociation (ECD) available in a custom-built ESI FTICR mass spectrometer. For linear permethylated malto-oligosaccharides, both CAD and "hot" ECD produced glycosidic cleavages (B, Y, C, and Z ions), cross-ring cleavages (A- and X-type), and internal cleavages (B/Y and C/Y type) to provide sequence and linkage information. For the branched N-linked glycans, CAD and "hot" ECD provided complementary structural information. CAD generated abundant B and Y fragment ions by glycosidic cleavages, whereas "hot" ECD produced dominant C and Z ions. A-type cross-ring cleavages were present in CAD spectra. Complementary A- and X-type cross-ring fragmentation pairs were generated by "hot" ECD, and these delineated the branching patterns and linkage positions. For example, 0, 4An and 3, 5An ions defined the linkage position of the major branch as the 6-position of the central core mannose residue. The internal fragments observed in CAD were more numerous and abundant than in "hot" ECD spectra. Since the triply charged (sodiated) molecular ion of the permethylated disialylated-biantennary N-linked glycan has relatively high abundance, it was isolated and fragmented in a "hot" ECD experiment and extensive fragment ions (glycosidic and complementary pairs of cross-ring cleavages) were generated to fully confirm the sequence, branching, and linkage assignments for this glycan.     

Characterization of amino acid side chain losses in electron capture dissociation

We have used electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry to characterize amino acid side chain losses observed during electron capture dissociation (ECD) of ten 7- to 14-mer peptides. Side-chain cleavages were observed for arginine, histidine, asparagine or glutamine, methionine, and lysine residues. All peptides containing an arginine, histidine, asparagine or glutamine showed the losses associated with that residue. Methionine side-chain loss was observed for doubly-protonated bombesin. Lysine side-chain loss was observed for triply-protonated dynorphin A fragment 1-13 but not for the doubly-protonated ion. The proximity of arginine to a methoxy C-terminal group significantly enhances the extent of side-chain fragmentation. Fragment ions associated with side-chain losses were comparable in abundance to those resulting from backbone cleavage in all cases. In the ECD spectrum of one peptide, the major product was due to fragmentation within an arginine side chain. Our results suggest that cleavages within side chains should be taken into account in analysis of ECD mass spectral data. Losses from arginine, histidine, and asparigine/glutamine can be used to ascertain their presence, as in the analysis of unknown peptides, particularly those with non-linear structures.     

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.     

Characterization of permethylated β-cyclodextrin-peptide noncovalently bound complexes using electron capture dissociation mass spectrometry (ECD MS)

Electron capture dissociation mass spectrometry (ECD MS) was carried out for a number of β-permethylated cyclodextrin (CD)-peptide noncovalent complexes in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Examined peptides included Angiotensin II (DRVYIHPF), Substance P (RPKPQQFFGLM), and Bradykinin (RPPGFSPFR) and its analogs (PPGFSPFR and RPPGFSPF). ECD MS for doubly protonated complexes [M:CD+2H]²⁺ mainly yielded cleavage of the backbones of the constituent peptide with little disassembly of a peptide and β-CD. Analysis of ECD MS fragments indicated that a protonated basic amino-acid residue or N-terminal amino group interacted more favorably with β-CD than did aromatic group-containing amino-acid residues (inclusion complex). In contrast to the formation of inclusion CD complexes in solution, we observed no specific evidence from our ECD MS mass spectra to support the generation of phenyl inclusion complexes in the gas phase. For gas-phase peptides, we suggest that ion–dipole interaction is the main driving force for the formation of noncovalent β-CD complexes rather than phenyl inclusion interactions.     

Detection of the reduced forms of radical adducts on the ESR trace using HPLC‐electrochemical detector‐ultraviolet absorption detector‐electron spin resonance‐MS

To detect and identify the electron spin resonance (ESR) silent forms of the α‐(4‐pyridyl‐1‐oxide)‐N‐tert‐butylnitrone (4‐POBN) radical adducts, an electrochemical detector (ECD) was employed as a reactor in the HPLC‐ECD‐UV absorption detector‐ESR‐MS (HPLC‐ECD‐UV‐ESR‐MS). The ECD was employed to regenerate the radical forms from the reduced forms. The reduced forms of the 4‐POBN/pentyl radical adducts were analyzed using the HPLC‐ECD‐UV‐ESR‐MS. On addition of the ECD applied potential of +0.3 V, a peak appeared on the ESR trace of the HPLC‐ECD‐UV‐ESR‐MS analyses, indicating that the radical forms are regenerated from the reduced forms. The HPLC‐ECD‐UV‐ESR‐MS analyses were also performed for the reaction mixtures of phenylhydrazine with CuCl₂. Two peaks (peaks I and II) were detected on the UV trace (300 nm) of the HPLC‐ECD‐UV‐ESR‐MS. The mass spectra showed that the peak I and peak II compounds are radical and reduced forms of the 4‐POBN/phenyl radical adducts under the ECD applied potential of 0.0 V. Peak I was only detected on the ESR trace under the ECD applied potential of 0.0 V. In addition to peak I, peak II appeared on the ESR trace under the ECD applied potential of +0.3 V, indicating that the reduced forms are oxidized to the corresponding radical forms.     

Structural characterization of the GM1 ganglioside by infrared multiphoton dissociation, electron capture dissociation, and electron detachment dissociation electrospray ionization FT-ICR MS/MS

Gangliosides play important biological roles and structural characterization of both the carbohydrate and the lipid moieties is important. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), electron detachment dissociation (EDD), and infrared multiphoton dissociation (IRMPD) provide extensive fragmentation of the protonated and deprotonated GM1 ganglioside. ECD provides extensive structural information, including identification of both halves of the ceramide and cleavage of the acetyl moiety of the N-acetylated sugars. IRMPD provides similar glycan fragmentation but no cleavage of the acetyl moiety. Cleavage between the fatty acid and the long-chain base of the ceramide moiety is seen in negative-ion IRMPD but not in positive-ion IRMPD of GM1. Furthermore, this extent of fragmentation requires a range of laser powers, whereas all information is available from a single ECD experiment. However, stepwise fragmentation by IRMPD may be used to map the relative labilities for a series of cleavages. EDD provides the alternative of electron-induced fragmentation for negative ions with extensive fragmentation, but suffers from low efficiency as well as complication of data analysis by frequent loss of hydrogen atoms. We also show that analysis of MS/MS data for glycolipids is greatly simplified by classification of product ion masses to specific regions of the ganglioside based solely on mass defect graphical analysis.     

Solution or Gas Phase? Oxidation and Radical Formation in Electrospray Ionization Mass Spectrometry (ESI MS)

The relationship between one‐electron (e^?) oxidation processes and the formation of radical cations of endogenous and exogenous compounds in vivo is of considerable interest. This paper reports on the experiments that allow FTICR mass spectrometric (MS) detection of ion signals that are consistent with the formation of radical cations of caffeine (CA) and theophylline (TP) during electrospray ionization (ESI) in ESI FTICR MS and in on‐line electrochemistry (EC)/ESI FTICR MS in positive mode. Significantly, the signals of the radicals of CA^?+ and TP^?+can be enhanced by simple modifications of the operating conditions in ESI MS, facilitating investigations of radical formation and related reactions.     

Negative-ion electron capture dissociation: radical-driven fragmentation of charge-increased gaseous peptide anions

The generation of gaseous polyanions with a Coulomb barrier has attracted attention as exemplified by previous studies of fullerene dianions. However, this phenomenon has not been reported for biological anions. By contrast, electron attachment to multiply charged peptide and protein cations has seen a surge of interest due to the high utility for tandem mass spectrometry (MS/MS). Electron capture dissociation (ECD) and electron transfer dissociation (ETD) involve radical-driven fragmentation of charge-reduced peptide/protein cations to yield N-C(α) backbone bond cleavage, resulting in predictable c'/z(?)-type product ions without loss of labile post-translational modifications (PTMs). However, acidic peptides, e.g., with biologically important PTMs such as phosphorylation and sulfonation, are difficult to multiply charge in positive ion mode and show improved ionization in negative-ion mode. We found that peptide anions ([M - nH](n-), n ≥ 1) can capture electrons within a rather narrow energy range (~3.5-6.5 eV), resulting in charge-increased radical intermediates that undergo dissociation analogous to that in ECD/ETD. Gas-phase zwitterionic structures appear to play an important role in this novel MS/MS technique, negative-ion electron capture dissociation (niECD).     

Solution-phase deuterium/hydrogen exchange at a specific residue using nozzle-skimmer and electron capture dissociation mass spectrometry

Information about protein conformation can be obtained with hydrogen/deuterium exchange (HDX) mass spectrometry. The isotopic solution-phase exchange of specific amide hydrogen atoms can be followed using low-vacuum nozzle-skimmer collision-induced dissociation (CID). In this study, the nozzle-skimmer technique was complemented by electron capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The solution-phase exchange at a specific residue is monitored by comparing isotopic distributions of two consecutive b- or c-type ions. While nozzle-skimmer fragmentation takes place in the low-vacuum region of the mass spectrometer, ECD occurs at ultra-high vacuum within the mass analyzer cell of the FTICR mass spectrometer. The dissociations take place at 10(-4) and 10(-9) mbar, respectively. Low-vacuum nozzle-skimmer fragmentation can result in intramolecular exchange between product ions and solvent molecules in the gas phase. Consequently, the solution-phase information about protein or peptide conformation is lost. It was not possible to monitor isotopic solution-phase exchange at the eighth residue in substance P, (Phe)8, with nozzle-skimmer CID. By using the in-cell ECD fragmentation method, the solution-phase exchange at the (Phe)8 residue was preserved during mass spectrometric analysis. This result shows the complementary aspects of applying fragmentation at low and at high vacuum, when studying isotopic exchange in solution at specific residues using FTICRMS.     

Electron capture dissociation of singly and multiply phosphorylated peptides

Analysis of phosphotyrosine and phosphoserine containing peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissociation (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissociation (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z(.)-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z(.) fragment ion products was observed in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatography combined with nano-electrospray FTMS/ECD facilitated phosphopeptide analysis and amino acid sequencing from crude proteolytic peptide mixtures.     

Electron Capture Dissociation of Disulfide, Sulfur–Selenium, and Diselenide Bound Peptides

To examine the electron capture dissociation (ECD) behavior of disulfide (S?CS), sulfur?Cselenium (S?CSe), and diselenide (Se?CSe) bonds-containing peptides, a series of free cysteine (Cys) and selenocysteine (Sec) containing peptides were reacted to form interchain S?CS, S?CSe, and Se?CSe bonds, and then studied using ECD with Fourier transform ion cyclotron mass spectrometry (FTICR MS). These results demonstrate that the radical has higher tendency to stay at selenium rather than sulfur after the cleavage of Se?CS bonds by ECD. In addition, ?CSH (?C33), ?CS (?C32), and ?CS + H (?C31) small neutral losses were all observed from the cleavage of C?CS bonds of a disulfide bound peptide. Similar, but minor, fragments were also detected in S?CSe bound peptides. In contrast, the cleavage of C?CSe bonds of the Se?CSe species mainly forms fragments with neutral loss of ?CSe + H (?C78.90868), and the radical tends to stay on the selenium of its corresponding complementary pair. Although the electron affinities of S atom (2.07?eV) and Se atom (2.02?eV) are very close; they have very different reactivity towards electrons. The replacement of sulfur with selenium greatly increases the electron affinities of S?CSe and Se?CSe bonds comparing to S?CS bonds (with an increase of electron affinity by about 0.20?eV by replacing a sulfur with a selenium) (Int J Quantum Chem 110:513-523, 2010), which in turn leads to different ECD fragmentation behavior and mechanisms. Our results are in good agreement with previously published ab initio calculations on Se?CSe compounds by other groups.     

Trace determination of alpha- and beta-endosulfan and three metabolites in human serum by gas chromatography electron capture detection and gas chromatography tandem mass spectrometry

Endosufan, alpha and beta, and three conversion products, sulphate, ether and lactone, were simultaneously determined in human serum by means of an analytical procedure which combines extraction with organic solvents, clean-up with H(2)SO(4) and by liquid column chromatography, and detection by gas chromatography (GC) using electron capture detection (ECD) and tandem mass spectrometry (MS/MS). The procedure was validated and the values of some merit figures, such as linear range, detection and quantitation limits, accuracy, precision and recovery, obtained with the GC/ECD and the GC/MS/MS methods, were compared. The lower limits of detection in GC/ECD and GC/MS/MS were 0.03 and 0.05 microg I(-1), respectively. The recovery of the pesticides at the 20 microg I(-1) concentration level was 60-65%, with the exception of endosufan alpha. Recovery studies at higher levels (100 and 200 microg I(-1)) were independent of pesticide concentration in serum samples. The application of the proposed analytical methodology to the determination of endosulfans and their metabolites in real samples was tested by analyzing serum samples from a population living in agricultural areas of Almeria (Spain). The results show the advantage of MS/MS over the ECD detector in the analysis of serum samples where matrix interferences can be confused with target pesticides.     

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.