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.     

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.     

Electron detachment dissociation for top-down mass spectrometry of acidic proteins

Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72% for Melittin, which lacks carboxylic acid functionalities, to 19% for an acidic 147-residue protein, to 12% for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO(2). Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins.     

IRMPD and ECD fragmentation of intermolecular cross-linked peptides

Despite the increasing number of studies using mass spectrometry for three dimensional analyses of proteins (MS3D), the identification of cross-linked peptides remains a bottleneck of the method. One of the main reasons for this is the lack of knowledge about the fragmentation of these species. Intermolecular cross-linked peptides are considered the most informative species present in MS3D experiment, since different peptides are connected by a cross-linker, the peptides chain can be either from a single protein, providing information about protein folding, or from two different proteins in a complex, providing information about binding partners, complex topology and interaction sites. These species tend to be large and highly charged in ESI, making comprehensive fragmentation by CID MS/MS problematic. On the other hand, these highly charged peptides are very suitable for dissociation using both infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD). Herein, we report the fragmentation study of intermolecular cross-linked peptides using IRMPD and ECD. Using synthetic peptides and different commercial cross-linkers, a series of intermolecular cross-linked peptides were generate, and subsequently fragmented by IRMPD and ECD in a FT-ICR-MS instrument. Due to the high mass accuracy and resolution of the FT-ICR, the fragment ions could be attributed with high confidence. The peptides sequence coverage and fragmentation features obtained from IRMPD and ECD were compared for all charge states.     

Free radical-induced site-specific peptide cleavage in the gas phase: Low-energy collision-induced dissociation in ESI- and MALDI mass spectrometry

Protein identification is routinely accomplished by peptide sequencing using mass spectrometry (MS) after enzymatic digestion. Site-specific chemical modification may improve peptide ionization efficiency or sequence coverage in mass spectrometry. We report herein that amino group of lysine residue in peptides can be selectively modified by reaction with a peroxycarbonate and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID) in electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) MS. Selective modification of lysine residue in peptides by our strategy can induce specific peptide cleavage at or near the lysine site. Studies using deuterated analogues of modified lysine indicate that fragmentation of the modified peptides involves apparent free-radical processes that lead to peptide chain fragmentation and side-chain loss. The formation of a-, c-, or z-types of ions in MS is reminiscent of the proposed free-radical mechanisms in low-energy electron capture dissociation (ECD) processes that may have better sequence coverage than that of the conventional CID method. This site-specific cleavage of peptides by free radical- promoted processes is feasible and such strategies may aid the protein sequencing analysis and have potential applications in top-down proteomics.     

Novel Cysteine Tags for the Sequencing of Non-Tryptic Disulfide Peptides of Anurans: ESI-MS Study of Fragmentation Efficiency

Mass spectrometry faces considerable difficulties in de novo sequencing of long non-tryptic peptides with S–S bonds. Long disulfide-containing peptides brevinins 1E and 2Ec from frog Rana ridibunda were reduced and alkylated with nine novel and three known derivatizing agents. Eight of the novel reagents are maleimide derivatives. Modified samples were subjected to MS/MS studies on FT-ICR and Orbitrap mass spectrometers using CAD/HCD or ECD/ETD techniques. Procedures, fragmentation patterns, and sequence coverage for two peptides modified with 12 tags are described. ECD/ETD and CAD fragmentation revealed complementary sequence information. Higher-energy collisionally activated dissociation (HCD) sufficiently enhanced y-ions formation for brevinin 1E, but not for brevinin 2Ec. Some novel tags [N-benzylmaleimide, N-(2,6-dimethylphenyl)maleimide] along with known N-phenylmaleimide and iodoacetic acid showed high total sequence coverage taking into account combined ETD and HCD fragmentation. Moreover, modification of long (34 residues) brevinin 2Ec with N-benzylmaleimide or N-(2,6-dimethylphenyl)maleimide yielded high sequence coverage and full C-terminal sequence determination with ECD alone.     

Liquid chromatography electron capture dissociation tandem mass spectrometry (LC-ECD-MS/MS) versus liquid chromatography collision-induced dissociation tandem mass spectrometry (LC-CID-MS/MS) for the identification of proteins

Electron capture dissociation (ECD) offers many advantages over the more traditional fragmentation techniques for the analysis of peptides and proteins, although the question remains: How suitable is ECD for incorporation within proteomic strategies for the identification of proteins? Here, we compare LC-ECD-MS/MS and LC-CID-MS/MS as techniques for the identification of proteins. Experiments were performed on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin, lysozyme, cytochrome c, alcohol dehydrogenase, and β-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides. The results show that although protein coverage was lower for LC-ECD-MS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags, providing greater confidence in protein assignment.     

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.     

The effect of phosphorylation on the electron capture dissociation of peptide ions

The effect of site and frequency of phosphorylation on the electron capture dissociation of peptide ions has been investigated. The ECD of a suite of synthetic peptides (APLSFRGSLPKSYVK; one unmodified, three singly-phosphorylated, three-doubly phosphorylated, and one triply-phosphorylated); two tryptic phosphopeptides (YKVPQLEIVPN(p)SAEER, alpha-casein and FQ(p)SEEQQQTEDELQDK, beta-casein) and their unmodified counterparts, were determined over a range of ECD cathode potentials. The results show that, for doubly-charged precursor ions, the presence of phosphorylation has a deleterious effect on ECD sequence coverage. The fragmentation patterns observed suggest that for peptides with multiple basic residues, the phospho-groups exist in their deprotonated form and form salt-bridges with protonated amino acid side chains. The fragmentation observed for the acidic tryptic peptides suggested the presence of noncovalent interactions, which were perturbed on phosphorylation. Increasing the ECD electron energy significantly improves sequence coverage. Alternatively, improved sequence coverage can be achieved by performing ECD on triply-charged precursor ions. The findings are important for the understanding of gas-phase fragmentation of phosphopeptides.     

New aspects in fragmentation of peptide nucleic acids: comparison of positive and negative ions by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The use of peptide nucleic acids (PNAs) is steadily increasing in biochemistry and diagnostics. So far, PNAs have mostly been investigated using cationic conditions in mass spectrometry. Furthermore, the use of fragmentation techniques developed for peptides and proteins like infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) has barely been examined. However, especially the fragmentation behavior of PNA oligomers in negative ion mode is of high importance, due to the ability to interact with nucleic acids which are almost exclusively analyzed in the negatively charged state. In the current study PNA fragmentations under cationic and anionic conditions were investigated and different fragmentation techniques like collision‐induced dissociation (CID), IRMPD and ECD were applied. Especially when using CID and IRMPD, amide bonds were broken, whereas ECD resulted in the elimination of nucleobases. Differences were also observed between positive and negative ionization, while the sequence coverage for the negative ions was superior to positive ions. The fragmentation behavior using IRMPD led to almost complete sequence coverage. Additionally, in anions the interesting effect of multiple eliminations of HNCO was found. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of Fragmentation Pathways of Peptide Modified with Quaternary Ammonium and Phosphonium Group as Ionization Enhancers

Peptide modification by a quaternary ammonium group containing a permanent positive charge is a promising method of increasing the ionization efficiency of the analyzed compounds, making ultra-sensitive detection even at the attomolar level possible. Charge-derivatized peptides may undergo both charge remote (ChR) and charge-directed (ChD) fragmentation. A series of model peptide conjugates derivatized with N,N,N-triethyloammonium (TEA), 1-azoniabicyclo[2.2.2]octane (ABCO), 2,4,6-triphenylopyridinium (TPP) and tris(2,4,6-trimetoxyphenylo)phosphonium (TMPP) groups were analyzed by their fragmentation pathways both in collision-induced dissociation (CID) and electron-capture dissociation (ECD) mode. The effect of the fixed-charge tag type and peptide sequence on the fragmentation pathways was investigated. We found that the aspartic acid effect plays a crucial role in the CID fragmentation of TPP and TEA peptide conjugates whereas it was not resolved for the peptides derivatized with the phosphonium group. ECD spectra are mostly dominated by c_n ions. ECD fragmentation of TMPP-modified peptides results in the formation of intense fragments derived from this fixed-charge tag, which may serve as reporter ion.     

Electron capture dissociation,electron detachment dissociation,and collision-induced dissociation of polyamidoamine (PAMAM) dendrimer ions with amino,amidoethanol, and sodium carboxylate surface groups

Here, we investigate the effect of the structure (generation) and nature of the surface groups of different polyamidoamine (PAMAM) dendrimers on electron-mediated dissociation, either electron capture dissociation (ECD) or electron detachment dissociation (EDD), and compare the fragmentation with that observed in collision-induced dissociation (CID). ECD and EDD of the PAMAM dendrimers resulted in simple mass spectra, which are straightforward to interpret, whereas CID produced complex mass spectra. The results show that electron-mediated dissociation (ECD and EDD) of PAMAM dendrimers does not depend on the nature of the surface group but tends to occur within the innermost generations. CID of the PAMAM dendrimers showed a strong dependence on the nature of the surface group and occurred mostly in the outer generation. The results demonstrate the potential utility of ECD and EDD as a tool for the structural analysis of PAMAM dendrimers.     

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).     

Evaluation of low energy CID and ECD fragmentation behavior of mono-oxidized thio-ether bonds in peptides

Thio-ether bonds in the cysteinyl side chain of peptides, formed with the most commonly used cysteine blocking reagent iodoacetamide, after conversion to sulfoxide, releases a neutral fragment mass in a low-energy MS/MS experiment in the gas phase of the mass spectrometer [6]. In this study, we show that the neutral loss fragments produced from the mono-oxidized thio-ether bonds (sulfoxide) in peptides, formed by alkyl halide or double-bond containing cysteine blocking reagents are different under low-energy MS/MS conditions. We have evaluated the low-energy fragmentation patterns of mono-oxidized modified peptides with different cysteine blocking reagents, such as iodoacetamide, 3-maleimidopropionic acid, and 4-vinylpyridine using FTICR-MS. We propose that the mechanisms of gas-phase fragmentation of mono-oxidized thio-ether bonds in the side chain of peptides, formed by iodoacetamide and double-bond containing cysteine blocking reagents, maleimide and vinylpyridine, are different because of the availability of acidic beta-hydrogens in these compounds. Moreover, we investigated the fragmentation characteristics of mono-oxidized thio-ether bonds within the peptide sequence to develop novel mass-spectrometry identifiable chemical cross-linkers. This methionine type of oxidized thio-ether bond within the peptide sequence did not show anticipated low-energy fragmentation. Electron capture dissociation (ECD) of the side chain thio-ether bond containing oxidized peptides was also studied. ECD spectra of the oxidized peptides showed a greater extent of peptide backbone cleavage, compared with CID spectra. This fragmentation information is critical to researchers for accurate data analysis of this undesired modification in proteomics research, as well as other methods that may utilize sulfoxide derivatives.     

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).     

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.     

Top-down mass spectrometry for sequencing of larger (up to 61 nt) RNA by CAD and EDD

We have studied the effect of solution additives on hydrolysis and charge state distribution in ESI MS of RNA. Lower and higher charge state ions can be electrosprayed from solutions containing 25 mM piperidine/25 mM imidazole and 1% vol. triethylamine, respectively, with base-catalyzed hydrolysis rates that are sufficiently slow to perform MS/MS experiments. These lower and higher charge state ions are suitable as precursors for CAD and EDD, respectively. We demonstrate nearly complete sequence coverage for 61 nt RNA dissociated by CAD, and 34 nt RNA dissociated by EDD, and suggest a mechanism for backbone fragmentation in EDD of RNA.     

Identification of single and double sites of phosphorylation by ECD FT-ICR/MS in peptides related to the phosphorylation site domain of the myristoylated alanine-rich c kinase protein

A series of phosphorylated test peptides was studied by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS). The extensive ECD-induced fragmentation made identification of phosphorylation sites for these peptides straightforward. The site(s) of initial phosphorylation of a synthetic peptide with a sequence identical to that of the phosphorylation site domain (PSD) of the myristoylated alanine-rich C kinase (MARCKS) protein was then determined. Despite success in analyzing fragmentation of the smaller test peptides, a unique site on the PSD for the first step of phosphorylation could not be identified because the phosphorylation reaction produced a heterogeneous mixture of products. Some molecules were phosphorylated on the serine closest to the N-terminus, and others on one of the two serines closest to the C-terminus of the peptide. Although no definitive evidence for phosphorylation on either of the remaining two serines in the PSD was found, modification there could not be ruled out by the ECD fragmentation data.     

Tandem mass spectrometric sequence characterization of synthetic thymidine-rich oligonucleotides

Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic oligonucleotide drugs, including modified oligonucleotides. Multiple factors can affect oligonucleotide MS/MS sequencing, including the intrinsic properties of oligonucleotides (i.e., nucleotide composition and structural modifications) and instrument parameters associated with the ion activation for fragmentation. In this study, MS/MS sequencing of a thymidine (T)-rich and phosphorothioate (PS)-modified DNA oligonucleotide was investigated using two fragmentation techniques: trap-type collision-induced dissociation (“CID”) and beam-type CID also termed as higher-energy collisional dissociation (“HCD”), preceded by a hydrophilic interaction liquid chromatography (HILIC) separation. A low to moderate charge state (−4), which predominated under the optimized HILIC-MS conditions, was selected as the precursor ion for MS/MS analysis. Comparison of the two distinctive ion activation mechanisms on the same precursor demonstrated that HCD was superior to CID in promoting higher sequence coverage and analytical sensitivity in sequence elucidation of T-rich DNA oligonucleotides. Specifically, HCD provided more sequence-defining fragments with higher fragment intensities than CID. Furthermore, the direct comparison between unmodified and PS-modified DNA oligonucleotides demonstrated a loss of MS/MS fragmentation efficiency by PS modification in both CID and HCD approaches, and a resultant reduction in sequence coverage. The deficiency in PS DNA sequence coverage observed with single collision energy HCD, however, was partially recovered by applying HCD with multiple collision energies. Collectively, this work demonstrated that HCD is advantageous to MS/MS sequencing of T-rich PS-modified DNA oligonucleotides.     

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.