Electron capture and collisionally activated dissociation mass spectrometry of doubly charged hyperbranched polyesteramides

Electron capture dissociation (ECD) of doubly protonated hyperbranched polyesteramide oligomers (1100-1900 Da) was examined and compared with the structural information obtained by low energy collisionally activated dissociation (CAD). Both the ester and amide bonds of the protonated species were cleaved easily upon ECD with the formation of odd electron (OE(.+)) or even electron (EE(+)) fragment ions. Several mechanistic schemes are proposed that describe the complex ECD fragmentation behavior of the multiply charged oligomers. In contrast to studies of biomolecules, the present results indicate that consecutive cleavages induced by intramolecular H-shifts are significant for ECD and of less importance for low energy CAD. The capture of an electron by the ionized species results in fragmentation associated with a redistribution of the excess internal energy over the products and the subsequent bond cleavage. Low energy, multiple collision CAD is found to be a more selective dissociation method than ECD in view of the observation that only amide bonds are cleaved for most of the hyperbranched polymers examined with CAD in this study. ECD appears not to provide complementary structural information compared to CAD in the study of hyperbranched polymers, even though a significantly more complex ECD fragmentation behavior is observed. ECD is shown to be of use for the structural characterization of large oligomers that may not dissociate upon low energy CAD. This is a direct result of the fact that ECD produces ionized hyperbranched oligomers with a relatively high internal energy.     

Charge/radical site initiation versus coulombic repulsion for cleavage of multiply charged ions. Charge solvation in poly(alkene glycol) ions

Electrospray ionization of poly(ethylene glycol) (PEG) followed by separation with Fourier-transform mass spectrometry traps (PEG100 + nH)n+ ions. Both collisionally activated dissociation (CAD) and electron capture dissociation (ECD) of these ions (n = 5, 6, 7) produce PEGx fragment ions in which the x values correspond closely to those for an equal distribution of charges in the linear polymer ion, e.g., for n = 7, near x = 1, 17, 34, 50, 67, 83, and 100. However, positions intermediate between these charges should represent the maximum coulombic repulsion, so this is not a specific driving force for fragmentation, which is instead consistent with charge site (CAD) or radical site (ECD) initiation. These conclusions were confirmed by studies of a variety of other poly(alkene glycol) polymers. For these, the ECD spectra of the protonated species are consistent with the predicted charge solvation by the ion's oxygen atoms.     

Comparison of electron capture dissociation and collisionally activated dissociation of polycations of peptide nucleic acids

Electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance mass spectrometry coupled with electrospray ionization enhances the sequence elucidation of peptide nucleic acids compared with conventional low-energy collisionally activated dissociation (CAD). Examples are shown where ECD produced complete or extensive sequence coverage in PNAs six to ten nucleobases long. However, facile base losses from the reduced species and low abundances of backbone ECD fragments presented a significant problem. This was rationalized through the lower degree of charge solvation on the backbone compared to polypeptides. Combination of both CAD and ECD data is advantageous, as these techniques produce cleavages at different sites.     

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.     

Collision-activated dissociation,infrared multiphoton dissociation,and electron capture dissociation of the <Emphasis Type="Italic">Bacillus anthracis</Emphasis> siderophore petrobactin and its metal ion complexes

Siderophores are high-affinity iron-chelating ligands produced by microorganisms to scavenge vital Fe(3+) from the environment. Thus, siderophores constitute potential therapeutic targets and their structural determination is important for exploiting their therapeutic value. Here, the virulence-associated siderophore petrobactin from Bacillus anthracis was characterized with electron capture dissociation (ECD). Fragmentation of doubly protonated petrobactin was investigated and compared to sustained off-resonance irradiation collision-activated dissociation (SORI CAD) and infrared multiphoton dissociation (IRMPD) of both the singly and doubly protonated species. These experiments demonstrate that ECD provides additional information (complementary bond cleavages) on the structure of petrobactin compared to both SORI CAD and IRMPD. Furthermore, complexes of petrobactin with divalent (Ca(2+), Fe(2+), and Co(2+)) and trivalent (Fe(3+) and Ga(3+)) metal cations were also subjected to SORI CAD and ECD. Again, most structural information was obtained from the ECD spectra. However, significant differences were found in both SORI CAD and ECD of metal complexes, dependent on the nature of the metal ion. Intriguingly, unique behavior, consistent with a recently proposed solution-phase structure, was observed for the highly preferred Fe(3+)-petrobactin complex.     

Preparation of Polyamide‐6 Submicrometer‐Sized Spheres by In Situ Polymerization

Polyamide‐6 (PA6) submicron‐sized spheres are prepared by two steps: (1) anionic ring‐opening polymerization of ε‐caprolactam in the presence of poly(ethylene glycol)‐block‐poly‐(propylene glycol)‐block‐poly(ethylene glycol)(PEG‐b‐PPG‐b‐PEG) and (2) separation of PA6 spheres by dissolving PEG‐b‐PPG‐b‐PEG from the prepared blends. The PA6 microspheres obtained are regular spherical, with diameter ranging from 200 nm to 2 μm and narrow size distribution, as confirmed by scanning electron microscopy. By comparison with PA6/PS and PA6/PEG systems, it is denominated that the PEG blocks in PEG‐b‐PPG‐b‐PEG can effectively reduce the surface tension of PA6 droplets and further decrease the diameter of the PA6 microspheres. The PPG block in PEG‐b‐PPG‐b‐PEG can prevent the PA6 droplets coalescing with each other, and isolated spherical particles can be obtained finally. The phase inversion of the PA6/PEG‐b‐PPG‐b‐PEG blends occurs at very low PEG‐b‐PPG‐b‐PEG content; the PEG‐b‐PPG‐b‐PEG phase can be removed by water easily. The whole experiment can be finished in a short time (approximately in half an hour) without using any organic solvents; it is an efficient strategy for the preparation of submicron‐sized PA6 microspheres.

     

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/transfer versus collisionally activated/induced dissociations: Solo or duet?

New ion fragmentation technologies--electron capture dissociation (ECD) and electron-transfer dissociation (ETD)--are based on interaction of multiply charged polypeptides with either free electrons (ECD) or anionic species (ETD). After initial difficulties, these ECD/ETD (ExD) technologies are now being increasingly implemented in high-throughput proteomics work. This critical analysis presents arguments for the combined use of ExD with the conventional low-energy collisional excitation CID/CAD (CxD). It is argued that the database search, a key technology in MS/MS-based proteomics, is vulnerable with respect to the incomplete sequence information obtainable with either of the techniques, peptide MS/MS homology being a major complicating factor. De novo sequencing is viewed as the only adequate answer to this challenge and it can be achieved only with combined use of ExD and CxD. The payoff in the form of additional sequence information is projected to exceed the costs of such implementation. The greatest impact of combining ExD and CxD is expected in high-resolution instruments.     

A simple method to estimate relative stabilities of polyethers cationized by alkali metal ions

Dissociation of doubly cationized polyethers, namely [P + 2X]²⁺ into [P + X]⁺ and X⁺, where P = polyethylene glycol (PEG), polypropylene glycol (PPG) and polytetrahydrofuran (PTHF) and X = Na, K and Cs, was studied by means of energy‐dependent collision‐induced dissociation tandem mass spectrometry. It was observed that the collision voltage necessary to obtain 50% fragmentation (CV₅₀) determined for the doubly cationized polyethers of higher degree of polymerization varied linearly with the number of degrees of freedom (DOF) values. This observation allowed us to correlate these slopes with the corresponding relative gas‐phase dissociation energies for binding of alkali ions to polyethers. The relative dissociation energies determined from the corresponding slopes were found to decrease in the order Na⁺ > K⁺ > Cs⁺ for each polyether studied, and an order PPG ≈ PEG > PTHF can be established for each alkali metal ion. Copyright © 2011 John Wiley & Sons, Ltd.     

Fragmentation of oligoribonucleotides from gas-phase ion-electron reactions

We have recently demonstrated that both electron capture dissociation (ECD) and electron detachment dissociation (EDD) can provide complementary sequence-specific cleavage of DNA compared with collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD). However, EDD is preferred because of more extensive fragmentation at higher sensitivity (due to its negative ion mode operation). Here, we extend the radical ion chemistry of these two gas-phase ion-electron reaction techniques to the characterization of RNA. Compared with DNA, rather limited information is currently available on the gas-phase fragmentation of RNA. We found that the ECD fragmentation patterns of the oligoribonucleotides A6, C6, and CGGGGC are nucleobase dependent, suggesting that cleavage proceeds following electron capture at the nucleobases. Only limited backbone cleavage was observed in ECD. EDD, on the other hand, provided complete sequence coverage for the RNAs A6, C6, G6, U6, CGGGGC, and GCAUAC. The EDD fragmentation patterns were different from those observed with CAD and IRMPD in that the dominant product ions correspond to d- and w-type ions rather than c- and y-type ions. The minimum differences between oligoribonucleotides suggest that EDD proceeds following direct electron detachment from the phosphate backbone.     

Study of Products of the Alkaline Decomposition of Hydrolysis Lignin by Atmospheric Pressure Photoionization High-Resolution Mass Spectrometry

A method of high resolution-mass spectrometry with acetone doped atmospheric pressure photoionization was used to study products of the alkaline solvolysis of hydrolysis lignin. It was found that the mass spectrum of the depolymerization products of hydrolysis lignin consists of about seven thousand peaks of oligomers, containing up to 10 aromatic units with an average molecular weight of 150 Da. Calculations of the elemental compositions of all detectable oligomers and their visualization on the van Krevelen coordinates allowed us to show that the studied sample differs from native (virgin released) lignin by the presence of fractions with high oxygen contents and highly unsaturated condensed structures, including polynuclear aromatic hydrocarbons. The structural units of lignin oligomers were characterized using an approach based on the collision induced dissociation of precursor ions in a broad m/z range.     

Native MS and ECD Characterization of a Fab–Antigen Complex May Facilitate Crystallization for X-ray Diffraction

Native mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)–VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis.

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The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for <Emphasis Type="Italic">O</Emphasis>-glycosylated and <Emphasis Type="Italic">O</Emphasis>-phosphorylated peptides

Electron capture dissociation (ECD) has become an alternative method to collision-activated dissociation (CAD) to avoid gas-phase cleavage of post-translational modifications carried by side chains from the peptide backbone. Nonetheless, as illustrated herein by the study of O-glycosylated and O-phosphorylated peptides, the extent of ECD fragmentations may be insufficient to cover the entire peptide sequence and to localize accurately these modifications. The present work demonstrates that the derivatization of peptides at their N-terminus by a phosphonium group improves dramatically and systematically the sequence coverage deduced from the ECD spectrum for both O-glycosylated and O-phosphorylated peptides compared with their native counterparts. The exclusive presence of N-terminal fragments (c-type ions) in the ECD spectra of doubly charged molecular cations simplifies peptide sequence interpretation. Thus, the combination of ECD and fixed charge derivatization appears as an efficient analytical tool for the extensive sequencing of peptides bearing labile groups.     

Size-selective protein adsorption to polystyrene surfaces by self-assembled grafted poly(ethylene glycols) with varied chain lengths

We report about the surface modification of polystyrene (PSt) with photoreactive alpha-4-azidobenzoyl-omega-methoxy poly(ethylene glycol)s (ABMPEG) of three different molecular weights (MWs of approximately 2, approximately 5, and approximately 10 kg/mol) and with two poly(ethylene glycol)/poly(propylene glycol) triblock copolymers (PEG-PPG-PEG) of about identical PEG/PPG ratio (80/20, w/w) and MW(PEG) of approximately 3 and approximately 6 kg/mol, all via adsorption from aqueous solutions. For ABMPEGs, an additional UV irradiation was used for photografting to the PSt. Contact angle (CA) and atomic force microscopy data revealed pronounced differences of the hydrophilicity/hydrophobicity and topography of the surfaces as a function of PEG type and concentration used for the modification. In all cases, an incomplete coverage of the PSt was observed even after modification at the highest solution concentrations (10 g/L). However, clear differences were seen between PEG-PPG-PEGs and ABMPEGs; only for the latter was a nanoscale-ordered interphase structure with an influence of MW(PEG) on the PEG density observed; after modification at the same solution concentrations, the density was significantly higher for lower MW(PEG). The adsorption of three proteins, myoglobin (Mgb), bovine serum albumin (BSA), and fibrinogen to the various surfaces was analyzed by surface plasmon resonance. Pronounced differences between the two PEG types with respect to the reduction of protein adsorption were found. At high, but still incomplete, surface coverage and similar CA, the shielding of ABMPEG layers toward the adsorption of Mgb and BSA was much more efficient; e.g., the adsorbed Mgb mass relative to that of unmodified PSt was reduced to 10% for ABMPEG 2 kg/mol while for both PEG-PPG-PEGs the Mgb mass was still around 100%. In addition, for the ABMPEG layers an effect of MW(PEG) on adsorbed protein mass-decrease with decreasing MW-could be confirmed; and the highest Mgb/BSA selectivities were also observed. A "two-dimensional molecular sieving", based on PEG molecules having a nanoscale order at the hydrophobic substrate polymer surface has been proposed, and the main prerequisites were the use of PEG conjugates which are suitable for an "end-on" grafting (e.g., ABMPEGs), the use of suitable (not too high) concentrations for the surface modification via adsorption/self-assembly, optionally the photografting on the substrate (possible only for ABMPEG), and presumably, a washing step to remove the excess of unbound PEGs. The results of this study also strongly support the hypothesis that the biocompatibility of hydrophobic materials can be very much improved by PEG modifications at surface coverages that are incomplete but have an ordered layer structure controlled by the size and steric interactions of surface-bound PEGs.     

Structural Heterogeneity of Doubly-Charged Peptide b-Ions

Performing collisionally activated dissociation (CAD) and electron capture dissociation (ECD) in tandem has shown great promise in providing comprehensive sequence information that was otherwise unobtainable by using either fragmentation method alone or in duet. However, the general applicability of this MS³ approach in peptide sequencing may be undermined by the formation of non-direct sequence ions, as sometimes observed under CAD, particularly when multiple stages of CAD are involved. In this study, varied-sized doubly-charged b-ions from three tachykinin peptides were investigated by ECD. Sequence scrambling was observed in ECD of all b-ions from neurokinin A (HKTDSFVGLM-NH₂), suggesting the presence of N- and C-termini linked macro-cyclic conformers. On the contrary, none of the b-ions from eledoisin (pEPSKDAFIGLM-NH₂) produced non-direct sequence ions under ECD, as it does not contain a free N-terminal amino group. ECD of several b-ions from Substance P (RPKPQQFFGLM-NH₂) showed series of c_m-Lys fragment ions which suggested that the macro-cyclic structure may also be formed by connecting the C-terminal carbonyl group and the ε-amino group of the lysine side chain. Theoretical investigation of selected Substance P b-ions revealed several low energy conformers, including both linear oxazolones and macro-ring structures, in corroboration with the experimental observation. This study showed that a b-ion may exist as a mixture of several forms, with their propensities influenced by its N-terminus, length, and certain side-chain groups. Further, the presence of several macro-cyclic structures may result in erroneous sequence assignment when the combined CAD and ECD methods are used in peptide sequencing.     

Interaction of sodium dodecyl sulfate with methacrylate-PEG comb copolymers

A series of sodium methacrylate and poly(ethylene glycol) (PEG) comb copolymers (MAA/PEG) with approximate PEG chain lengths of 7, 11, and 22 ethylene oxide units were synthesized by free radical polymerization. Their weight-average molecular mass was found to be approximately 66 000. A commercial sample of a PEG comb polymer with an acrylic backbone was also used in the studies (Sokalan HP 80). The interaction of the MAA/PEG comb polymers and pure sodium methacrylate (SPMA) with sodium dodecyl sulfate (SDS) was studied by ESR spectroscopy using 5-doxyl stearic acid (5-DSA) spin probe and by conductivity measurements. Surfactant aggregation in water occurred at SDS concentrations lower than the surfactant critical micelle concentration (cmc) and depended on the polymer concentration. The observations have been attributed to changes in the effective ionic strength of the systems due to the polymer itself, and it has been concluded that there is no interaction between the MAA/PEG comb copolymers or SPMA and SDS. This has been confirmed by the fact that the decrease in surfactant aggregation concentration is similar in magnitude to the decrease observed on adding NaCl when counterion ion condensation effects are taken into account. It is apparent that the electrostatic repulsions between the surfactant molecules and the methacrylate backbone of the MAA/PEG comb copolymers inhibit association of SDS with the PEG side chains.     

Quantitative comparison of a corona-charged aerosol detector and an evaporative light-scattering detector for the analysis of a synthetic polymer by supercritical fluid chromatography

A corona-charged aerosol detector (CAD) was developed to improve the sensitivity, reproducibility and quantitativeness of detection as compared to evaporative light-scattering detector (ELSD) for liquid chromatography. Our laboratory used the corona CAD as a detector for supercritical fluid chromatography (SFC) and evaluated its performance compared to the ELSD by using a certified reference material of poly(ethylene glycol) (PEG) and a well-defined equimass mixture of uniform PEG oligomers. The corona CAD was able to detect a 10 times more dilute solution of uniform oligomers compared to the ELSD. Although the original data of molecular mass by ELSD was 4.6% smaller than the certified value of PEG 1000, molecular mass distribution obtained by corona CAD was virtually almost the same as the certified value without any calibrations.     

The rapid characterisation of poly(ethylene glycol) oligomers using desorption electrospray ionisation tandem mass spectrometry combined with novel product ion peak assignment software

A rapid method for the characterisation of polyglycol esters and ethers is described which uses accurate mass desorption electrospray ionisation (DESI) quadrupole time-of-flight mass spectrometry (Q-ToFMS). The results are combined with newly developed software which aids the interpretation of product ions produced using collision-induced dissociation (CID) of selected precursor ions. The poly(ethylene glycol) (PEG) samples analysed were PEG dibenzoate, PEG monooleate, PEG butyl ether, PEG bis(2-ethyl hexanoate) and PEG diacrylate. Lithium metal was used for cationisation of the PEG oligomers since it yielded the most useful structural information compared with other group I metals. The full scan mass spectra and product ion mass spectra were all obtained in <5 s. Interpretation of the MS/MS product ion spectra, using the product ion interpretation software which incorporates previously developed fragmentation rules, was carried out in <1 s.     

Electron detachment dissociation of neutral and sialylated oligosaccharides

Electron detachment dissociation (EDD) has recently been shown by Amster and coworkers to constitute a valuable analytical approach for structural characterization of glycosaminoglycans. Here, we extend the application of EDD to neutral and sialylated oligosaccharides. Both branched and linear structures are examined, to determine whether branching has an effect on EDD fragmentation behavior. EDD spectra are compared to collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) spectra of the doubly and singly deprotonated species. Our results demonstrate that EDD of both neutral and sialylated oligosaccharides provides structural information that is complementary to that obtained from both CAD and IRMPD. In all cases, EDD resulted in additional cross-ring cleavages. In most cases, cross-ring fragmentation obtained by EDD is more extensive than that obtained from IRMPD or CAD. Our results also indicate that branching does not affect EDD fragmentation, contrary to what has been observed for electron capture dissociation (ECD).     

Liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS): an early overview

Fourier-transform ion cyclotron resonance mass spectrometry has developed into one of the most powerful analytical techniques. This unique technique enables acquisition of high-resolution mass spectra with high accuracy, which in turn enables determination of the elemental composition of the analyzed compounds. Coupling with liquid chromatography affords a separation technique with a high-resolution detector which can be used to investigate very complex matrices. In this review some important instrumental developments are described and applications are presented; these show the advantages and disadvantages of this combination.Abbreviations CAD Collision-activated dissociation - CZE Capillary zone electrophoresis - ECD Electron-capture dissociation - FT Fourier-transform - MS Mass spectrometry - ICR Ion cyclotron resonance - IRMPD Infrared multi-photon dissociation - LC Liquid chromatography - LSIMS Liquid secondary-ion mass spectrometry - SORI Sustained off-resonance irradiation This contribution is dedicated to Professor Dr M.T. Reetz on the occasion of his 60th birthday.