Sustained Off-resonance Irradiation Collision-activated Dissociation Research Articles

Electron capture dissociation of divalent metal-adducted sulfated oligosaccharides

κ-Carrageenan sulfated oligosaccharides containing one, two, and four sulfate groups were complexed with divalent metal cations and electrosprayed in positive ion mode. The resulting metal complexes were subjected to sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) and electron capture dissociation (ECD). The presence of divalent metal cations in the electrospray solution facilitates formation of doubly positively charged precursor ions in the gas phase. Unsurprisingly, abundant sulfate group (SO 3) loss is observed in SORI CAD of sulfated oligosaccharides complexed with divalent metal cations. By contrast, sulfation is retained in ECD of metal-sulfated oligosaccharide complexes, providing information on the site of sulfation. Most product ions resulting from ECD are C/Z- or B/Y-type ions, corresponding to cleavage of glycosidic bonds linking two sugar units. However, several A/X-type product ions corresponding to sugar cross-ring cleavage were also observed in ECD. Such cross-ring fragments are important in carbohydrate analysis because they provide linkage information.

Differentiation of Isomeric Hydrocarbons by Using [ClMn(H2O)]+Chemical Ionization and Collision-Activated Dissociation in a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Chemical ionization (CI) using the [ClMn(H2O)]+ reagent ion combined with collision-activated dissociation is demonstrated to allow the differentiation of isomeric alkanes (C8H18 and C9H20) and alkenes (C8H16) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The [ClMn(H2O)]+ ion reacts with each isomeric hydrocarbon molecule to produce a water replacement product [hence providing molecular weight (MW) information], with no accompanying fragmentation. Differentiation of the isomers is achieved on the basis of the different fragmentation patterns observed upon sustained off-resonance irradiation collision-activated dissociation (SORI−CAD) of the isolated water replacement products. Upon activation, the weak bond of the hydrocarbon to ClMn+ is broken. ClMn+ is the major fragment ion for linear alkanes. However, for branched alkanes and all alkenes, the leaving ClMn+ abstracts a weakly bound hydride, an alkyl anion, or an alkene from the hydrocarbon to generate diagnostic carbocation...

Ultraviolet photodissociation at 266 nm of phosphorylated peptide cations

Ultraviolet (UV) photodissociation (PD) experiments using 266 nm light were performed for a series of phosphopeptide cations in a Fourier transform mass spectrometer. The objective of the experiments was to determine whether 266 nm UV irradiation on the phosphopeptide cations would induce unique peptide backbone dissociation. In addition, the general behavior of the phosphate loss (-80 or -98 Da) was monitored, particularly for those phosphopeptides with a phosphotyrosine residue that itself is a UV chromophore. For phosphopeptides with a UV chromophore, their photodissociation behavior was very similar to that of low-energy sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD), with a few exceptions. For example, b- and y-type peptide backbone fragments were prevalent, and their dephosphorylation behavior was consistent with that of the SORI-CAD results. For phosphoserine peptides, the loss of a phosphate group was always observed. On the other hand, for phosphotyrosine peptides, the phosphate loss was found to be dependent on the presence of a basic amino group in the sequence and the charge state of the precursor ions, in agreement with the CAD results in the literature. However, hydrogen atom loss or aromatic side chain loss, which is known to be the excited state specific fragmentation pathway, was rarely observed in our 266 nm UV PD experiments, in contrast to the previous UV PD literature (particularly at 220 nm). The mechanism for these observations is described in terms of dominant internal conversion followed by intramolecular vibrational energy redistribution (IVR).

H/D Exchange Kinetics: Experimental Evidence for Formation of Different b Fragment Ion Conformers/Isomers During the Gas-Phase Peptide Sequencing

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) combined with H/D exchange reactions was utilized to explore the existence of different b(5)(+) and b(4)(+) fragment ion conformers/isomers of hexapeptide WHWLQL in the gas phase. Distinct H/D exchange trends for protonated WHWLQL ([M + H](+)) and its b(5)(+) and b(4)(+) fragment ions (with ND(3)) were observed. Isolated (12)C(all) isotopomers of both b(5)(+) and b(4)(+) fragment ions yielded bimodal distributions of H/D exchanged product ions. The H/D exchange reaction kinetics also confirmed that b(5)(+) and b(4)(+) fragment ions exist as combination of slow-exchanging ("s") and fast-exchanging ("f") species. The calculated rate constant for the first labile hydrogen exchange of [M + H](+) (k([M + H](+)) = 3.80 +/- 0.7 x 10(-10) cm(3) mol(-1) s(-1)) was approximately 30 and approximately 5 times greater than those for the "s" and "f" species of b(5)(+), respectively. Data from H/D exchange of isolated "s" species at longer ND(3) reaction times confirmed the existence of different conformers or isomers for b(5)(+) fragment ions. The sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) of WHWLQL combined with the H/D exchange reactions indicate that "s" and "f" species of b(5)(+) and b(4)(+) fragment ions can be produced in the ICR cell as well as the ESI source. The significance of these observations for detailed understanding of protein sequencing and ion fragmentation pathways is discussed.

ESI‐FTICRMS Characterization of the Intermediates in Cycloisomerization of Trifluoromethyl‐substituted Enynols through Changing the Protecting Group of Substrate

AbstractA chemical method of changing the protecting group was coupled to MS, deuterium‐labeled and NMR methods to characterize the intermediates in the palladium(II)‐catalyzed cycloisomerization of trifluoromethyl‐substituted enynols. It was demonstrated that the method of changing the protecting group of substrates not only caused a decrease in reaction speed, but also provided long‐lived reaction intermediates detectable by conventional spectroscopic techniques. In this sense, two assumed reaction intermediates were intercepted and detected by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FTICRMS). For further characterization of these intermediates, the gas‐phase behavior of these intermediates was studied by sustained off‐resonance irradiation collision‐activated dissociation (SORI‐CAD) experiments. On the basis of the detailed, spectral interpretation of the palladium‐containing species, the fragmentation pathways of the intermediates have been proposed. The deuterium‐labeled experiment helped confirm the nature of the intermediates.

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.

Ion–molecule reactions of trimethylborate allow the mass spectrometric identification and counting of functional groups in protonated bifunctional oxygen-containing compounds and polyols

A mass spectrometric method has been developed for the identification of functional groups in unknown bifunctional oxygen-containing compounds and for the identification and counting of the hydroxyl groups in polyols. This method utilizes gas-phase ion–molecule reactions of protonated analytes with neutral trimethylborate (TMB) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The diagnostic reaction sequence involves proton abstraction from the protonated analyte by TMB, followed by the addition of the analyte to TMB and elimination of methanol. The functional groups in bifunctional oxygen-containing compounds are identified based on the total number of TMB molecules that have added to the protonated analyte and/or the number of methanol molecules lost, or by sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) of the reaction products. The number of hydroxyl groups in polyols is revealed by the number of methanol molecules lost during their reactions with TMB. Reactions of protonated carboxylic acids with TMB also lead to elimination of a methanol molecule. However, carboxylic acids can be differentiated from the isomeric hydroxyketones based on the loss of HO-B(OCH 3) 2 upon SORI-CAD of the reaction product. Reactions of protonated amides with TMB also lead to elimination of a methanol molecule. However, amides can be differentiated from bifunctional oxygen-containing compounds based on the loss of O B R from the reaction product upon SORI-CAD. Protonated amines do not react with TMB.

Collision-Activated Dissociation, Infrared Multiphoton Dissociation, and Electron Capture Dissociation of the Bacillus anthracis 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.

Electron Capture Dissociation as Structural Probe for Noncovalent Gas-Phase Protein Assemblies

Electron capture dissociation (ECD) of proteins in Fourier transform ion cyclotron resonance mass spectrometry usually leads to charge reduction and backbone-bond cleavage, thereby mostly retaining labile, intramolecular noncovalent interactions. In this report, we evaluate ECD of the 84-kDa noncovalent heptameric gp31 complex and compare this with sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) of the same protein. Unexpectedly, the 21+ charge state of the gp31 oligomer exhibits a main ECD pathway resulting in a hexamer and monomer, disrupting labile, intermolecular noncovalent bonds and leaving the backbone intact. Unexpectedly, the charge separation over the two products is highly proportional to molecular weight. This indicates that a major charge redistribution over the subunits of the complex does not take place during ECD, in contrast to the behavior observed when using SORI-CAD. We speculate that the ejected monomer retains more of its original structure in ECD, when compared to SORI-CAD. ECD of lower charge states of gp31 does not lead to dissociation of noncovalent bonds. We hypothesize that the initial gas-phase structure of the 21+ charge state is significantly different from the lower charge states. These structural differences result in the different reaction pathways when using ECD.

ESI-FTICR-MS Studies on Gas Phase Fragmentation Reactions of ArPd(PPh 3) 2I Complexes

Gas-phase fragmentation reactions of [ArPd(PPh3)2]+ were studied by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The results of sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) experiments provide detailed insights into mechanisms for the gas-phase fragmentation reactions of these complex ions. The PC bond cleavage mediated by palladium is investigated in the gas phase. There are two competitive fragmentation pathways for the complex ions [ArPd(PPh3)2]+ (Ar = p-OCH3-C6H4, p-CH3-C6H4, p-tBu-C6H4, p-NH2-C6H4, p-COCH3-C6H4, and p-F-C6H4) of electron-donating and electron-withdrawing aromatic iodides. Path A proceeds through reductive elimination of [ArPd(PPh3)2]+ to produce the product ion [PPh3Ar]+. Path B mostly proceeds via phenyl migration from the triphenylphosphine ligand to the palladium center by cleavage of the phosphorus-phenyl bond to give a palladium-phenyl intermediate, and subsequent reductive elimination of the intermediate to yield a product ion [PPh4]+. The result of deuterium-labeling experiments provides evidence for the phenyl shift between the palladium center and the coordinated ligand through cleavage of the PC bond. The complex ions [(o-CH3-C6H4)Pd(PPh3)2]+, [(o-2,6-Me2-C6H3)Pd(PPh3)2]+, and [(C10H7)Pd(PPh3)2]+ display more fragmentation pathways, two of which are similar to those of the ions [ArPd(PPh3)2]+ (Ar = p-OCH3-C6H4, p-CH3-C6H4, p-tBu-C6H4, p-NH2-C6H4, p-COCH3-C6H4, p-F-C6H4), and the third pathway involves loss of one molecule of benzene and one PPh3 ligand. The electronic effect and steric effect of the aryl groups also exhibit different influences on the fragmentation pathways.

Direct Screening of a Dynamic Combinatorial Library Using Mass Spectrometry

A dynamic combinatorial library (DCL) screening approach is described that permits direct identification of the effective (from ineffective) combination of building blocks in the equilibrating DCL. The approach uses Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) together with sustained off-resonance irradiation collision activated dissociation (SORI-CAD) to detect noncovalent protein-DCL ligand complexes under native conditions. It was shown that in a single, rapid experiment one could concurrently identify all the ligands of interest from the DCL against a background of inactive DCL ligands while still in the presence of the target protein. This result has demonstrated that mass spectrometry may provide a fast preliminary screening approach to identify DCL candidates for later verification with more traditional but time-consuming analysis. The MS/MS enables DCL mixtures to be effectively deconvoluted without the need for either chromatography, synthesis of DCL sub-libraries, conversion of the DCL to a static library, or disruption of the protein-ligand complexes before analysis--all typically necessary for the current screening method for DCLs.

Studies of gas‐phase fragmentation reactions of [Pd(PPh3)2(OCOR)]+ by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Gas-phase fragmentation reactions of [Pd(PPh3)2(OCOR)]+ (R = H, CH3, CD3, C2H5, n-C3H7, n-C6H13 and C6H5) were studied by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS). In sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) experiments, the complexes all dissociated to yield the same product ion at m/z 629.1. We propose that the fragmentation pathway occurs through the elimination of RCOOH and a palladium(IV) hydride intermediate. Semi-empirical (PM3) calculations shed light on the mechanism for the fragmentation reactions of these compounds. The results of deuterium-labeling experiments indicate that the protons of RCOOH lost from [Pd(PPh3)2(OCR)]+ originate from the phenyl in the triphenylphosphine ligand. [Pd(PPh3)2(OCOH)]+ undergoes two competitive pathways in SORI-CAD experiments, one of which is similar to that of [Pd(PPh3)2(OCOR)]+ (R = CH3, CD3, C2H5, n-C3H7, n-C6H13 and C6H5), and the other involves decarboxylation. The present study demonstrates that MS could play an important role in studying the gas-phase chemistry of palladium hydrides.

Multipole-Storage-Assisted Dissociation for the Characterization of Large Proteins and Simple Protein Mixtures by ESI-FTICR-MS

In Fourier transform ion cyclotron resonance mass spectrometry, collisionally activated dissociation (CAD) typically is accomplished within the analyzer ion cell. An alternative approach of multipole-storage-assisted dissociation (MSAD) has previously been demonstrated by inducing collisional fragmentation in the external multipole that is usually employed for ion accumulation. To explore the utility of MSAD for interrogating intact proteins and simple protein mixtures in a multiplexed manner, we have investigated the means of controlling the collisional energy and the fragmentation pattern for this experimental approach. With protein samples in the low micromolar concentration range, the two major experimental parameters affecting MSAD in the hexapole region were found to be the dc offset voltage and accumulation time. While low-energy MSAD of intact proteins yields fragment ions similar to sustained off resonance irradiation collision-activated dissociation (SORI-CAD), high-energy MSAD induces sequential fragmentation for intact proteins to yield a rich variety of singly charged ions in the m/z 600-1200 Da region. Each of the seven proteins (Mr range of 8.5-116 kDa) examined in this study exhibited their own characteristic MSAD fragmentation pattern, which could be used as a signature of the presence of a given protein, even in a mixture. In addition, any MSAD fragment can be isolated and dissociated further by SORI-CAD in an MS3-type experiment inside the FTICR analyzer cell. This presents a novel way to interrogate the identities of these fragment ions as well as obtain amino acid sequence tag information that can be used to identify proteins from mixtures.

Characterization of an N-acylated glucagon-like peptide-1 derivative by electron capture dissociation

An N-acylated glucagon-like peptide 1 derivative was characterized by Fourier transform ion cyclotron resonance mass spectrometry. Both electron capture dissociation (ECD) and sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) were employed. While ECD revealed full sequence coverage, site of modification, branching point, structure of the palmitoylated modification, SORI-CAD produced less complete and more ambiguous information attributable to facile losses of the fatty acid group from both parent and fragments. Thus, ECD showed a superior characterization performance over SORI-CAD in analysis of N-acylated polypeptides.

Tandem mass spectrometryfor the determination of the sitesof DNA interstrand cross-link

Formation of DNA interstrand cross-link is implicated in the mechanism of anticancer activity of some drugs. Here we examined the fragmentation of deprotonated ions of double-stranded oligodeoxynucleotides (ODNs) that are covalently held together with either a mitomycin C or a 4,5',8-trimethylpsoralen. Our results showed that, upon collisional activation, the covalently-bound duplex ODNs cleaved to give a series of wn and [an-base] ions; the sites of interstrand cross-linking could be determined from the mass shifts of some product ions. In addition, compared with the product-ion spectra acquired on an ion trap, those obtained from sustained off-resonance irradiation-collisionally activated dissociation (SORI-CAD) on a Fourier transform mass spectrometer offered high mass-resolving power, which facilitated unambiguous assignment of product ions and made it an effective method for locating the cross-linking sites.

Applications of Electron−Ion Dissociation Reactions for Analysis of Polycationic Chitooligosaccharides in Fourier Transform Mass Spectrometry

Singly protonated, doubly protonated, and sodiated pentaglucosamide (GlcNAc)(5), oligoglucosamines (GlcN)(m)(), and (GlcN)(3)GlcN(3OH14:0) were analyzed in an FTICR mass spectrometer by electron-ion dissociation reactions and compared to collision activation. The general fragmentation mode was found as the asymmetrical sequence fragments (B(n)() and minor C(n)() ion series) with full sequence coverage. Molecular mass information of each glucosamide or glucosamine residue can be readily obtained from the ion series. Fragmentation by electron capture dissociation revealed additional fragmentation of the N-acetyl moiety compared to sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) and electron-induced dissociation (EID). Sodiated GlcNAc(5) molecular adduct ions were analyzed by EID and compared to CAD. Both techniques provided full sequence coverage. EID was more effective, but CAD resulted in the cross-ring ion products (0,2)A(n)() and (2,4)A(n)() for all relevant glucosamide residues.

Characterization of Monomeric and Dimeric Forms of Recombinant Sml1p-histag Protein by Electrospray Mass Spectrometry

Sml1p is small protein that binds to and inhibits the activity of ribonucleotide reductase (RNR)33Abbreviations used: RNR, ribonucleotide reductase; ORF, open reading frame; ES-FTICR-MS, electrospray Fourier transform ion cyclotron resonance mass spectrometry; NTA, nitrilotriacetic acid; IOD, image optical density; SORI-CAD, sustained off-resonance irradiation collision-activated dissociation; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; DTT, dithiothreitol., a protein enzyme complex that controls the balance and level of the cellular deoxynucleotide diphosphate pools that are critical for DNA synthesis and repair. In this respect, Sml1p is a checkpoint protein whose function is to regulate the activity of the large subunit of RNR (Rnr1p). Sml1p is thought to be regulated by the MEC1/RAD53 cell cycle checkpoint pathway. Neither the structure of Sml1p nor its complex to Rnr1p is well known. In this report, we describe how a recombinant Sml1p-histag protein (in both monomeric and dimeric forms) can be characterized with electrospray mass spectrometry. Mass spectrometry can play a vital role in the study of the Sml1p-Rnr1p complex by: (1) confirming the identities and purities of recombinant proteins such as Sm1lp-histag (with mass accuracy and resolution far superior to SDS–PAGE) and (2) verifying the presence or absence of PTM, chemical modifications, or metal–ion binding to the protein species, which may alter the function and binding of the protein partners.

The Analysis of Conducting Polymers by Electrospray Fourier Transform Mass Spectrometry. Part I: Ionene Polymers

The analysis of synthetic 6-2, 6-3 and 6-4 ionene polymers by electrospray ionization Fourier transform mass spectrometry (ESIFTMS) is reported here. This is the first in a series of papers on the analysis of conducting polymers utilizing soft ionization techniques, thus allowing for the analysis of intact molecular ions. The syntheses of these individual polymers result in a variety of compounds with different molecular structures which are related to the normal linear ionene polymer. While the mechanisms by which the 6-2 and the 6-3 ionene polymers are synthesized are very similar, the synthesis of 6-4 ionene proceeds differently. In addition, the presence of a gas-phase bromide ion salt-bridged to the backbone of the polymer chain is observed in the resulting mass spectra, as well as the possibility of intramolecular cyclization. The latter has been proposed previously. This cyclization process becomes more possible as the number of methylene groups in the linking chain increases. Finally, we observed that the presence of a bromide ion in the sample helped the interpretation of sustained off-resonance irradiation collisionally-activated dissociation (SORI-CAD). Based on the isotopic pattern provided by the halogens, a “built-in” label which can be traced from the fragment ions back to the parent ion peak is found to be useful in structural assignments.

Simulation for internal energy deposition in sustained off-resonance irradiation collisional activation using a monte carlo method

This paper proposes a novel computational approach employing a Monte Carlo method, aimed at an improved understanding of the dynamics and energetics of activated ions in sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) experimental events of Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). In SORI-CAD events, internal energies of activated ions are complicatedly associated with their motion undergoing off-resonance excitation (i.e. alternate accelerations and decelerations) and inherently stochastic ion-neutral collisions. Several types of pseudo-random generators were adapted to probability density functions (PDFs) which characterize the ion-neutral collision process. Simulated ion trajectories involve the realistic feature of pressurized SORI-CAD events, such as a collisional damping and those which have not been illustrated in conventional analytical approaches. The proposed method can simulate the time-varying translational and internal energies of activated ions. The present result suggests that the internal energy of a SORI-activated ion should be inversely proportional to the cube of the SORI excitation frequency offset. Copyright 1999 John Wiley & Sons, Ltd.