Gas-phase dissociation pathways of a tetrameric protein complex

The gas-phase dissociation of the tetrameric complex transthyretin (TTR) has been investigated with tandem-mass spectrometry (tandem-MS) using a nanoflow-electrospray interface and a quadrupole time-of-flight (Q-TOF) mass spectrometer. The results show that highly charged monomeric product ions dissociate from the macromolecular complex to form trimeric products. Manipulating the pressure conditions within the mass spectrometer facilitates the formation of metastable ions. These were observed for the transitions from tetrameric to monomeric and trimeric product ions and additionally for losses of small molecules associated with the protein complex in the gas phase. These results are interpreted in the light of recent mechanisms for the electrospray process and provide insight into the composition and factors governing the stability of macromolecular ions in the gas phase.     

Metastable atom‐activated dissociation mass spectrometry: leucine/isoleucine differentiation and ring cleavage of proline residues

Extensive backbone fragmentation resulting in a‐, b‐, c‐, x‐, y‐ and z‐type ions is observed of singly and doubly charged peptide ions through their interaction with a high kinetic energy beam of argon or helium metastable atoms in a modified quadrupole ion trap mass spectrometer. The ability to determine phosphorylation‐sites confirms the observation with previous reports and we report the new ability to distinguish between leucine and isoleucine residues and the ability to cleave two covalent bonds of the proline ring resulting in a‐, b‐, x‐, y‐, z‐ and w‐type ions. The fragmentation spectra indicate that fragmentation occurs through nonergodic radical ion chemistry akin to electron capture dissociation (ECD), electron transfer dissociation (ETD) and electron ionization dissociation mechanisms. However, metastable atom‐activated dissociation mass spectrometry demonstrates three apparent benefits to ECD and ETD: (1) the ability to fragment singly charged precursor ions, (2) the ability to fragment negatively charged ions and (3) the ability to cleave the proline ring that requires the cleavage of two covalent bonds. Helium metastable atoms generated more fragment ions than argon metastable atoms for both substance P and bradykinin regardless of the precursor ion charge state. Reaction times less than 250 ms and efficiencies approaching 5% are compatible with on‐line fragmentation, as would be desirable for bottom‐up proteomics applications. Copyright © 2009 John Wiley & Sons, Ltd.     

Electron detachment dissociation of dermatan sulfate oligosaccharides

The structural characterization of glycosaminoglycans (GAG) oligosaccharides has been a long-standing challenge in the field of mass spectrometry. In this work, we present the application of electron detachment dissociation (EDD) Fourier transform mass spectrometry to the analysis of dermatan sulfate (DS) oligosaccharides up to 10 residues long. The EDD mass spectra of DS oligosaccharides were compared with their infrared multiphoton dissociation (IRMPD) mass spectra. EDD produces more abundant fragmentation than IRMPD with far less loss of SO3 from labile sulfate modifications. EDD cleaves all glycosidic bonds, yielding both conventional glycosidic bond fragmentation as well as satellite peaks resulting from the additional loss of 1 or 2 hydrogen atoms. EDD also yields more cross-ring fragmentation than IRMPD. For EDD, abundant cross-ring fragmentation in the form of A- and X-ions is observed, with 1,5Xn cleavages occurring for all IdoA residues and many of the GalNAc4S residues, except at the reducing and nonreducing ends. In contrast, IRMPD produces only A-type cross-ring fragmentation for long oligosaccharides (dp6-dp10). As all the structurally informative fragment ions observed by IRMPD appear as a subset of the peaks found in the EDD mass spectrum, EDD shows great potential for the characterization of GAG oligosaccharides using a single tandem mass spectrometry experiment.     

Electron capture dissociation mass spectrometry of metallo-supramolecular complexes

The electron capture dissociation (ECD) of metallo-supramolecular dinuclear triple-stranded helicate Fe₂L₃⁴⁺ ions was determined by Fourier transform ion cyclotron resonance mass spectrometry. Initial electron capture by the di-iron(II) triple helicate ions produces dinuclear double-stranded complexes analogous to those seen in solution with the monocationic metal centers Cu^I or Ag^I. The gas-phase fragmentation behavior [ECD, collision-induced dissociation (CID), and infrared multiphoton dissociation (IRMPD)] of the di-iron double-stranded complexes, (i.e., MS³ of the ECD product) was compared with the ECD, CID, and IRMPD of the Cu^I and Ag^I complexes generated from solution. The results suggest that iron-bound dimers may be of the form Fe₂^IL₂²⁺ and that ECD by metallo-complexes allows access, in the gas phase, to oxidation states and coordination chemistry that cannot be accessed in solution.     

Formation and dissociation processes of gas-phase detergent micelles

Growing interest in micelles to protect membrane complexes during the transition from solution to gas phase prompts a better understanding of their properties. We have used ion mobility mass spectrometry to separate and assign detergent clusters formed from the n-trimethylammonium bromide series of detergents. We show that cluster size is independent of detergent concentration in solution, increases with charge state, but surprisingly decreases with alkyl chain length. This relationship contradicts the thermodynamics of micelle formation in solution. However, the liquid drop model, which considers both the surface energy and charge, correlates extremely well with the experimental cluster size. To explore further the properties of gas-phase micelles, we have performed collision-induced dissociation on them during tandem mass spectrometry. We observed both sequential asymmetric charge separation and neutral evaporation from the precursor ion cluster. Interestingly, however, we also found markedly different dissociation pathways for the longer alkyl chain detergents, with significantly fewer intermediate ions formed than for those with a shorter alkyl chain. These experiments provide an essential foundation for understanding the process of the gas-phase analysis of membrane protein complexes. Moreover they imply valuable mechanistic details of the protection afforded to protein complexes by detergent clusters during gas-phase activation processes.     

Influence of charge state and sodium cationization on the electron detachment dissociation and infrared multiphoton dissociation of glycosaminoglycan oligosaccharides

Electron detachment dissociation (EDD) Fourier transform mass spectrometry has recently been shown to be a useful method for tandem mass spectrometry analysis of sulfated glycosaminoglycans (GAGs). EDD produces abundant glycosidic and cross-ring fragmentations that are useful for localizing sites of sulfation in GAG oligosaccharides. Although EDD fragmentation can be used to characterize GAGs in a single tandem mass spectrometry experiment, SO3 loss accompanies many peaks and complicates the resulting mass spectra. In this work we demonstrate the ability to significantly decrease SO3 loss by selection of the proper ionized state of GAG precursor ions. When the degree of ionization is greater than the number of sulfate groups in an oligosaccharide, a significant reduction in SO3 loss is observed in the EDD mass spectra. These data suggested that SO3 loss is reduced when an electron is detached from carboxylate groups instead of sulfate. Electron detachment occurs preferentially from carboxylate versus sulfate for thermodynamic reasons, provided that carboxylate is in its ionized state. Ionization of the carboxylate group is achieved by selecting the appropriate precursor ion charge state, or by the replacement of protons with sodium cations. Increasing the ionization state by sodium cation addition decreases, but does not eliminate, SO3 loss from infrared multiphoton dissociation of the same GAG precursor ions.     

Symmetrical gas-phase dissociation of noncovalent protein complexes via surface collisions

Previous gas-phase dissociation experiments of protein-protein complexes have resulted in product ion distributions that are asymmetric by charge and mass, providing limited insight into the chemical nature of subunit organization and interaction. In these experiments, a symmetric charge distribution results from an "energy sudden" collision of protein-protein complexes with a surface, indicating that it may be possible to probe the suboligomeric structure of noncovalent complexes in the gas phase. It is proposed that energy sudden surface activation of cytochrome C homodimers results in dissociation without significant unfolding of one of the monomeric subunits. Previously proposed mechanisms for the dissociation of protein-protein complexes are discussed in the context of these results. These experiments demonstrate the potential to preserve the structural details of subunit interaction within a protein-protein complex and help elucidate the asymmetric nature of macromolecular dissociation in the gas phase.     

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-induced dissociation of glycosaminoglycan tetrasaccharides

Electron detachment dissociation (EDD) Fourier transform mass spectrometry has recently been shown to be a powerful tool for examining the structural features of sulfated glycosaminoglycans (GAGs). The characteristics of GAG fragmentation by EDD include abundant cross-ring fragmentation primarily on hexuronic acid residues, cleavage of all glycosidic bonds, and the formation of even- and odd-electron product ions. GAG dissociation by EDD has been proposed to occur through the formation of an excited species that can undergo direct decomposition or ejects an electron and then undergoes dissociation. In this work, we perform electron-induced dissociation (EID) on singly charged GAGs to identify products that form via direct decomposition by eliminating the pathway of electron detachment. EID of GAG tetrasaccharides produces cleavage of all glycosidic bonds and abundant cross-ring fragmentation primarily on hexuronic acid residues, producing fragmentation similar to EDD of the same molecules, but distinctly different from the products of infrared multiphoton dissociation or collisionally activated decomposition. These results suggest that observed abundant fragmentation of hexuronic acid residues occurs as a result of their increased lability when they undergo electronic excitation. EID fragmentation of GAG tetrasaccharides results in both even- and odd-electron products. EID of heparan sulfate tetrasaccharide epimers produces identical fragmentation, in contrast to EDD, in which the epimers can be distinguished by their fragment ions. These data suggest that for EDD, electron detachment plays a significant role in distinguishing glucuronic acid from iduronic acid.     

Sequence analysis of peptide:oligonucleotide heteroconjugates by electron capture dissociation and electron transfer dissociation

Mass spectrometry analysis of protein-nucleic acid cross-links is challenging due to the dramatically different chemical properties of the two components. Identifying specific sites of attachment between proteins and nucleic acids requires methods that enable sequencing of both the peptide and oligonucleotide component of the heteroconjugate cross-link. While collision-induced dissociation (CID) has previously been used for sequencing such heteroconjugates, CID generates fragmentation along the phosphodiester backbone of the oligonucleotide preferentially. The result is a reduction in peptide fragmentation within the heteroconjugate. In this work, we have examined the effectiveness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) for sequencing heteroconjugates. Both methods were found to yield preferential fragmentation of the peptide component of a peptide:oligonucleotide heteroconjugate, with minimal differences in sequence coverage between these two electron-induced dissociation methods. Sequence coverage was found to increase with increasing charge state of the heteroconjugate, but decreases with increasing size of the oligonucleotide component. To overcome potential intermolecular interactions between the two components of the heteroconjugate, supplemental activation with ETD was explored. The addition of a supplemental activation step was found to increase peptide sequence coverage over ETD alone, suggesting that electrostatic interactions between the peptide and oligonucleotide components are one limiting factor in sequence coverage by these two approaches. These results show that ECD/ETD methods can be used for the tandem mass spectrometry sequencing of peptide:oligonucleotide heteroconjugates, and these methods are complementary to existing CID methods already used for sequencing of protein-nucleic acid cross-links.     

Surface-induced dissociation of small molecules, peptides, and non-covalent protein complexes

This article provides a perspective on collisions of ions with surfaces, including surface-induced dissociation (SID) and reactive ion scattering spectrometry (RISS). The content is organized into sections on surface-induced dissociation of small ions, surface characterization of organic thin films by collision of well-characterized ions into surfaces, the use of SID to probe peptide fragmentation, and the dissociation of large non-covalent complexes by SID. Examples are given from the literature with a focus on experiments from the authors' laboratory. The article is not a comprehensive review but is designed to provide the reader with an overview of the types of results possible by collisions of ions into surfaces.     

Photoinduced dissociation of heparin-derived oligosaccharides controlled by charge location

The development of strategies based on mass spectrometry to help for deep structural analysis of acidic oligosaccharides remains topical. We thus examined the dissociation behavior of deprotonated ions of heparin-derived di- to tetra-saccharides under UV irradiation at 220 nm. Depending on the ionization state of the carboxylic groups, an oxidized species issued from electron photodetachment was observed in complement to photoinduced fragmentation of precursor ions. The influence of the charge location in the oligosaccharide dianions on the balance between photodissociation and electron photodetachment is examined and a way to direct the relaxation pathways, (i.e., dissociation versus electron detachment), is proposed using sodium adducts. The oxidized species was subjected to activated-electron photodetachment (activated-EPD) leading to complementary informative fragment ions to those issued from photodissociation. Directed photoinduced dissociation at 220 nm and activated-EPD should complement the more conventional CAD and IRMPD activation modes for deeper structural analysis of acidic oligosaccharides-derived anions.     

Infrared multiphoton and collision-induced dissociation studies of some gaseous alkylamine ions

Collision-induced dissociation and infrared multiphoton dissociation of ions formed in di- and tri-ethylamine, di- and tri-n-propylamine, and di-isopropylamine were investigated by Fourier-transform ion-cyclotron resonance mass spectrometry. Molecular ions of all amines except di-n-propylamine produced similar fragment ions when subjected to either dissociation technique. The initial fragmentation involved C_αC_β bond cleavage, loss of an alkyl radical, and formation of an immonium ions. Subsequent fragmentations of the immonium ions produced by both dissociation mechanisms involved McLafferty-type rearrangements and loss of alkenes. The molecular ion of di-n-propylamine fragmented by a different mechanism when subjected to infrared irradiation. Protonated molecules of di- and tri-n-propylamine yielded C₃H₆ and an ammonium ion upon infrared multiphoton dissociation, while protonated molecules of the other amines did not dissociate when this technique was applied. In contrast, collision-induced dissociation produced fragmentation for all protonated molecules. Explanation of the different fragmentations observed for the two dissociation techniques is given in terms of a mechanism involving a tight transition state for protonated di- and tri-n-propylamine dissociation.     

Comparison of collisionally activated dissociation mass spectra for the identification of cyclopeptides and cyclodepsipeptides

Collisionally activated dissociation (CAD) spectra of protonated molecules of cyclopeptides and cyclodepsipeptides obtained with two different mass spectrometry systems were compared. Fragmentations were obtained either from collisions induced in the ion source of an electrospray mass spectrometer fitted with a single quadrupole by increasing the extracting cone voltage or from collisions with an inert gas in a free-field area of a fast-atom bombardment (FAB) mass spectrometer. Similar fragmentation pathways were produced with the two configurations even though actual tandem mass spectrometry experiments with magnetic and electric sectors provided more information than cone-induced dissociations. However, only the latter mode allowed us to perform mass spectrometric analyses coupled to liquid chromatography (LC/ESI-MS) at low cost on commercially widespread instruments. Copyright © 1999 John Wiley & Sons, Ltd.     

A novel tandem quadrupole mass spectrometer allowing gaseous collisional activation and surface induced dissociation.

A novel tandem quadrupole mass spectrometer is described that enables gaseous collision-induced dissociation (CID) and surface-induced dissociation (SID) experiments. The instrument consists of a commercially available triple quadrupole mass spectrometer connected to an SID region and an additional, orthogonal quadrupole mass analyser. The performance of the instrument was evaluated using leucine-enkephalin, allowing a comparison between CID and SID, and with previous reports of other SID instruments. The reproducibility of SID data was assessed by replicate determinations of the collision energy required for 50% dissociation of leucine-enkephalin; excellent precision was observed (standard deviation of 0.6 eV) though, unexpectedly, the reproducibility of the equivalent figure for CID was superior. Several peptides were analysed using SID in conjunction with liquid secondary-ion mass spectrometry or electrospray; a comparison of the fragmentation of singly protonated peptide ions and the further dissociation of y-type fragments was consistent with the equivalence of the latter fragments to protonated peptides. Few product ions attributable to high-energy cleavages of amino acid side-chains were observed. The SID properties were investigated of a series of peptides differing only in the derivatization of a cysteine residue; similar decomposition efficiencies were observed for all except the cysteic acid analogue, which demonstrated significantly more facile fragmentation.     

Energy-dependent collision-induced dissociation study of buprenorphine and its synthetic precursors

The collision-induced dissociation (CID) of protonated buprenorphine ([M+H](+) ) and four related compounds was studied by electrospray quadrupole/time-of-flight mass spectrometry (ESI-QTOF MS). The fragmentation pathways were investigated by using energy-dependent CID and pseudo-MS(3) (in-source CID combined with tandem mass spectrometry (MS/MS)) methods. The first steps of the fragmentation are the parallel losses of the substituents from the non-aromatic ring moieties. Depending on the applied collision energies, a large number of further fragment ions arising from the cross-ring cleavages of the core-ring structure were observed. Based on the experimental results, a generalized fragmentation scheme was developed for the five buprenorphine derivatives highlighting the differences for the alternatively substituted compounds. The collision-energy-dependent fragmentation profile of buprenorphine is visualized in a two-dimensional plot to aid its fingerprint identification.     

Characterisation of anthocyanidins by electrospray ionisation and collision-induced dissociation tandem mass spectrometry.

The present study describes the use of electrospray ionisation mass spectrometry, in combination with collision-induced dissociation (CID) and tandem mass spectrometry, for the structural characterisation of anthocyanidins and their O-glycosides. The high-energy CID spectra of [M-Cl](+) ions of the free aglycones show characteristic fragmentation pathways, which provide useful information about the substitution pattern in the A- and B-rings of each compound. The major fragmentation observed in the high-energy CID spectra of [M-Cl](+) ions of anthocyanins involves loss of the mono- or disaccharide units resulting in ions containing only the aglycone moiety. From the spectral data, the identity of the aglycone can be established as well as the number and the class of monosaccharide units in the O-glycosides.     

Qualitative characterization of biomolecular zinc complexes by collisionally induced dissociation

Nanospray and collisionally induced dissociation (CID) on a quadrupole/time-of-flight mass spectrometer were used to examine the complexes formed between the zinc ion binding protein metallothionein and a series of peptides related to glutathione. The objective of the study was to determine if CID could be used to distinguish complexes that are stabilized by co-chelation of a zinc ion from non-covalent complexes that were formed in some other way. Differences in the collision energy required for dissociation and, more importantly, differences in the distribution of zinc ions between the pairs of dissociation products suggest that mass spectrometry can provide qualitative information about the bimolecular chelation of metal ions. The potential application to zinc chelates is particularly important, since biological chelates do not provide signals directly detectable by NMR, M?ssbauer or other spectroscopies. The observations reported here also allowed a molecular mechanism to be proposed to explain the differences observed by others in the physiological interactions of reduced and oxidized glutathione with metallothionein.     

Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization

We applied electrospray ionization (ESI) tandem quadrupole mass spectrometry to establish the fragmentation pathways of ceramides under low energy collisional-activated dissociation (CAD) by studying more than thirty compounds in nine subclasses. The product-ion spectra of the [M + Li]+ ions of ceramides contain abundant fragment ions that identify the fatty acyl substituent and the long-chain base (LCB) of the molecules, and thus, the structure of ceramides can be easily determined. Fragment ions specific to each ceramide subclasses are also observed. These feature ions permit differentiation among different ceramide subclasses. The ion series arising from the classical C-C bond cleavages that were reported in the fast-atom bombardment (FAB)-high energy tandem mass spectrometry is not observable; however, the product-ion spectra contain multiple fragment ions informative for structural characterization and isomer identification. We also investigated the tandem mass spectra of the fragment ions generated by in-source CAD (pseudo-MS3) and of the deuterium-labeling molecular species obtained by H/D exchange to support the ion structure assignments and the proposed fragmentation pathways that lead to the ion formation.     

Collision-induced dissociation of cytidine and its derivatives

The collision-induced dissociation of adenosine, uridine and guanosine, and their corresponding nucleobases has been published previously.1-3 Here we report the collision-induced dissociation of cytidine and the elucidation of its fragmentation pathways using stable isotope-labeled cytidines, through a quadrupole ion trap for tandem mass spectrometry up to MS(4). Furthermore, we investigated the collision-induced dissociation of five cytidine derivatives: 3-methylcytidine, N(4)-methyl-2'-deoxycytidine, 5-methylcytidine, 2-thiocytidine and N(4)-acetylcytidine. The primary fragmentation pathway was the neutral loss of ribose. MS(3) on the retained nucleobase generally resulted in an intense signal from the elimination of ammonia, but also in fragment ions characteristic of the different cytosine derivatives. On the basis of the MS(n) data, fragmentation pathways and plausible mechanisms are suggested.