Matrix‐free detection of intact ions from proteins in argon‐cluster secondary ion mass spectrometry

In the secondary ion mass spectrometry (SIMS) of organic substances, the molecular weight of the intact ions currently detectable is at best only as high as 1000 Da, which for all practical purposes prevents the technique from being applied to biomaterials of higher mass. We have developed SIMS instrumentation in which the primary ions were argon cluster ions having a kinetic energy per atom, controlled down to 1 eV. On applying this instrumentation to several peptides and proteins, the signal intensity of fragment ions was decreased by a factor of 10² when the kinetic energy per atom was decreased below 5 eV; moreover, intact ions of insulin (molecular weight (MW): 5808) and cytochrome C (MW: 12 327) were detected without using any matrix. These results indicate that fragmentation can be substantially suppressed without sacrificing the sputter yield of intact ions when the kinetic energy per atom is decreased to the level of the target's dissociation energy. This principle is fully applicable to other biomolecules, and it can thus be expected to contribute to applications of SIMS to biomaterials in the future. Copyright © 2009 John Wiley & Sons, Ltd.     

Dissociation of heme from gaseous myoglobin ions studied by infrared multiphoton dissociation spectroscopy and Fourier-transform ion cyclotron resonance mass spectrometry

Detachment of heme prosthetic groups from gaseous myoglobin ions has been studied by collision-induced dissociation and infrared multiphoton dissociation in combination with Fourier-transform ion cyclotron resonance mass spectrometry. Multiply charged holomyoglobin ions (hMbn+) were generated by electrospray ionization and transferred to an ion cyclotron resonance cell, where the ions of interest were isolated and fragmented by either collision with Ar atoms or irradiation with 3 mum photons, producing apomyoglobin ions (aMbn+). Both charged heme loss (with [Fe(III)-heme]+ and aMb(n-1)+ as the products) and neutral heme loss (with [Fe(II)-heme] and aMbn+ as the products) were detected concurrently for hMbn+ produced from a myoglobin solution pretreated with reducing reagents. By reference to Ea = 0.9 eV determined by blackbody infrared radiative dissociation for charged heme loss of ferric hMbn+, an activation energy of 1.1 eV was deduced for neutral heme loss of ferrous hMbn+ with n = 9 and 10.     

Identification of a new fragment ion type in the collision-induced dissociation spectra of peptides: Formation of a2-16 ions

The identification of an ion observed in the high-energy collision-induced dissociation spectra of several model peptides is reported. The ion, observed at m/z 99 for the peptide pentaalanine (Ala₅) and designated a₂-16, is shown to have an elemental formula of C₅H₉NO by high-resolution peak matching. The precursor ion spectrum of the a₂-16 ion and product ion spectra of the a₂ and the a₂+ 1 ions for Ala₅ suggest that this ion is formed by the loss of 17 u (presumably NH₃) from the a₂+1 ion and, to a lesser extent, by loss of 28 u (presumably CO) from the b₂-16 ion. On the basis of the data presented and other experimental evidence, a structure and mechanism for the formation of the a₂-16 ion is proposed.     

Charge transfer dissociation of phosphocholines: gas‐phase ion/ion reactions between helium cations and phospholipid cations

Phospholipid cations formed by electrospray ionization were subjected to excitation and fragmentation by a beam of 6 keV helium cations in a process termed charge transfer dissociation (CTD). The resulting fragmentation pattern in CTD is different from that of conventional collision‐induced dissociation, but analogous to that of metastable atom‐activated dissociation and electron‐induced dissociation. Like collision‐induced dissociation, CTD yields product ions indicative of acyl chain lengths and degrees of unsaturation in the fatty acyl moieties but also provides additional structural diagnostic information, such as double bond position. Although CTD has not been tested on a larger lipid sample pool, the extent of structural information obtained demonstrates that CTD is a useful tool for lipid structure characterization, and a potentially useful tool in future lipidomics workflows. CTD is relatively unique in that it can produce a relatively strong series of 2+ product ions with enhanced abundance at the double bond position. The generally low signal‐to‐noise ratios and spectral complexity of CTD make it less appealing than OzID or other radical‐induced methods for the lipids studies here, but improvements in CTD efficiency could make CTD more appealing in the future. Copyright © 2017 John Wiley & Sons, Ltd.     

Surface-induced dissociation of molecular ions in a quadrupole ion trap mass spectrometer

A method is reported by which surface-induced dissociation is used to activate ions stored in a quadrupole ion trap mass spectrometer. The method employs a short (< 5 μs), fast-rising (< 20-ns rise time), high voltage direct current (dc) pulse, which is applied to the endcaps of a standard Paul-type quadrupole ion trap. This is in contrast to the application of an alternating current (ac) signal normally used to resonantly excite and dissociate ions in the trap. The effect of the dc pulse is to cause the ions rapidly to become unstable in the radial direction and subsequently to collide with the ring electrode. Sufficient internal energy is acquired in this collision to cause high energy fragmentations of relatively intractable molecular ions such as pyrene and benzene. The dissociations of limonene are used to demonstrate that high energy demand processes increase in relative importance in the dc pulse experiment compared with the usual resonance excitation method used to cause activation. The fragments are scanned out of the ion trap using the conventional mass-selective instability scan mode. Simulations of ion motion in the trap provide evidence that surface collisions occur at kinetic energies in the range of tens to several hundred electronvolts. The experiments also demonstrate that production of fragment ions is sensitive to the phase of the main radiofrequency drive voltage at the point when the dc is initiated.     

Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision‐induced dissociation

Migration of sulfate groups between hydroxyl groups was identified after collision‐induced dissociation (CID) of sulfated oligosaccharides in an ion trap mass spectrometer in negative ion mode. Analysis of various sulfated oligosaccharides showed that this was a common phenomenon and was particularly prominent in sulfated oligosaccharides also containing sialic acid. It was also shown that the level of migration was increased when the sulfate was positioned on the flexible areas of the oligosaccharides not involved in the pyranose ring, such as the extra‐cyclic C‐6 carbon of hexoses or N‐acetylhexosamines, or on reduced oligosaccharide. This suggested that migration is dependent on the spatial availability of the sulfate in the ion trap during collision. It is proposed that the migration is initiated when the negatively charged ‐SO₃^– residue attached to the oligosaccharide precursor becomes protonated by a CID‐induced proton transfer. This is supported by the CID fragmentation of precursor ions depleted of acidic protons such as doubly charged [M – 2H]^2– ions or the sodiated [M + Na – 2H]^– ions of oligosaccharides containing one sulfate and one sialic acid in the same molecule. Compared to the CID fragmentation of their monocharged [M – H]^– ions, no migration was observed in CID of proton depleted precursors. Alternative fragmentation parameters to suppress migration of sulfated oligosaccharides also showed that it was not present when sulfated oligosaccharides were fragmented by HCD (High‐Energy C‐trap Dissociation) in an Orbitrap mass spectrometer. Copyright © 2011 John Wiley & Sons, Ltd.     

Unexpected linear ion trap collision‐induced dissociation and Fourier transform ion cyclotron resonance infrared multi‐photon dissociation fragmentation of a hydrated C‐glycoside of 5‐fluorouridine formed by the action of the pseudouridine synthases RluA and TruB

As part of the investigation of the pseudouridine synthases, 5‐fluorouridine in RNA was employed as a mechanistic probe. The hydrated, rearranged product of 5‐fluorouridine was isolated as part of a dinucleotide and found to undergo unusual fragmentation during mass spectrometry, with the facile loss of HNCO from the product pyrimidine ring favored over phosphodiester bond rupture. Although the loss of HNCO from uridine and pseudouridine is well established, the pericyclic process leading to their fragmentation cannot operate with the saturated pyrimidine ring in the product of 5‐fluorouridine. Based on the MSⁿ results and calculations reported here, a new mechanism relying on the peculiar disposition of the functional groups of the product pyrimidine ring is proposed to account for the unusually facile fragmentation. Copyright © 2011 John Wiley & Sons, Ltd.     

Investigation of c ions formed by N‐terminally charged peptides upon collision‐induced dissociation

Peptide fragments such as b and y sequence ions generated upon low‐energy collision‐induced dissociation have been routinely used for tandem mass spectrometry (MS/MS)‐based peptide/protein identification. The underlying formation mechanisms have been studied extensively and described within the literature. As a result, the ‘mobile proton model’ and ‘pathways in competition model’ have been built to interpret a majority of peptide fragmentation behavior. However, unusual peptide fragments which involve unfamiliar fragmentation pathways or various rearrangement reactions occasionally appear in MS/MS spectra, resulting in confused MS/MS interpretations. In this work, a series of unfamiliar c ions are detected in MS/MS spectra of the model peptides having an N‐terminal Arg or deuterohemin group upon low‐energy collision‐induced dissociation process. Both the protonated Arg and deuterohemin group play an important role in retention of a positive charge at the N‐terminus that is remote from the cleavage sites. According to previous reports and our studies involving amino acid substitutions and hydrogen–deuterium exchange, we propose a McLafferty‐type rearrangement via charge‐remote fragmentation as the potential mechanism to explain the formation of c ions from precursor peptide ions or unconventional b ions. Density functional theory calculations are also employed in order to elucidate the proposed fragmentation mechanisms. Copyright © 2016 John Wiley & Sons, Ltd.     

Dissociation of peptide ions by fast atom bombardment in a quadrupole ion trap

A new technique for fragmentation of cations and anions of peptides stored in ion traps including radiofrequency devices is described. The technique involves irradiation of peptide ions by a beam of particles generated by a fast atom bombardment (FAB) gun. This irradiation leads to fragmentation of N--C(alpha) backbone bonds (c- and z-fragments) and S--S bonds for cations and C(alpha)-C backbone bonds (a- and x-fragments) for anions of peptides. The fragmentation patterns observed are hypothesized to be due to the interaction of peptide ions with metastable, electronically excited species generated by the FAB gun. Interaction of a metastable atom A* with a peptide n-cation M(n+) leads to the electron transfer from the metastable atom to the polycation through the formation of an ion-pair collision complex A(+.) . . . M((n-1)+.) and subsequent fragmentation of the peptide cation. Thus, for polycations, this metastable-induced dissociation of ions (MIDI) is similar to the phenomenon of electron capture dissociation (ECD). Interaction of A* with an anion leads to the deexcitation of the metastable species and detachment of an electron from the anion. This in turn leads to backbone fragmentation similar to that in electron detachment dissociation (EDD). The MIDI technique is robust and efficient, and it is applicable to peptides in as low charge states as 2+ or 2-.     

Comparison of in‐source collision‐induced dissociation and beam‐type collision‐induced dissociation of emerging synthetic drugs using a high‐resolution quadrupole time‐of‐flight mass spectrometer

In‐source collision‐induced dissociation (CID) is commonly used with single‐stage high‐resolution mass spectrometers to gather both a molecular formula and structural information through the collisional activation of analytes with residual background gas in the source region of the mass spectrometer. However, unlike tandem mass spectrometry, in‐source CID does not involve an isolation step prior to collisional activation leading to a product ion spectrum composed of fragment ions from any analyte present during the activation event. This work provides the first comparison of in‐source CID and beam‐type CID spectra of emerging synthetic drugs on the same instrument to understand the fragmentation differences between the two techniques and to contribute to the scientific foundations of in‐source CID. Electrospray ionization–quadrupole time‐of‐flight (ESI‐Q‐TOF) mass spectrometry was used to generate product ion spectra from in‐source CID and beam‐type CID for a series of well‐characterized fentanyl analogs and synthetic cathinones. A comparison between the fragmentation patterns and relative ion abundances for each technique was performed over a range of fragmentor offset voltages for in‐source CID and a range of collision energies for beam‐type CID. The results indicate that large fragmentor potentials for in‐source CID tend to favor higher energy fragmentation pathways that result in both kinetically favored pathways and consecutive neutral losses, both of which produce more abundant lower mass product ions relative to beam‐type CID. Although conditions can be found in which in‐source CID and beam‐type CID provide similar overall spectra, the in‐source CID spectra tend to contain elevated noise and additional chemical background peaks relative to beam‐type CID.     

Investigation of ionic liquids under Bi‐ion and Bi‐cluster ions bombardment by ToF‐SIMS

A systematic study of five different imidazolium‐based room temperature ionic liquids, 1‐butyl‐3‐methylimidazolium acetate, 1‐butyl‐3‐methylimidazolium nitrate, 1‐butyl‐3‐methylimidazolium iodide, 1‐butyl‐3‐methylimidazolium hexafluorophosphate and 1‐butyl‐3‐methylimidazolium bis(trifluoromethylsulfonyl)imide were carried out by means of time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) in positive and negative ion mode. The compounds were measured under Bi‐ion and Bi‐cluster ions (Bi_2–7⁺, Bi_{3, 5}²⁺) bombardment, and spectral information and general rules for the fragmentation pattern are presented. Evidence for hydrogen bonding, due to high molecular secondary cluster ions, could be found. Hydrogen bonding strength could be estimated by ToF‐SIMS via correlation of the anionic yield enhancement with solvent parameters. Copyright © 2010 John Wiley & Sons, Ltd.     

Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance

Fourier-transform ion cyclotron resonance instrumentation is uniquely applicable to an unusual new ion chemistry, electron capture dissociation (ECD). This causes nonergodic dissociation of far larger molecules (42 kDa) than previously observed (<1 kDa), with the resulting unimolecular ion chemistry also unique because it involves radical site reactions for similarly larger ions. ECD is highly complementary to the well known energetic methods for multiply charged ion dissociation, providing much more extensive protein sequence information, including the direct identification of N- versus C-terminal fragment ions. Because ECD only excites the molecule near the cleavage site, accompanying rearrangements are minimized. Counterintuitively, cleavage of backbone covalent bonds of protein ions is favored over that of noncovalent bonds; larger (>10 kDa) ions give far more extensive ECD if they are first thermally activated. This high specificity for covalent bond cleavage also makes ECD promising for studying the secondary and tertiary structure of gaseous protein ions caused by noncovalent bonding.     

Prompt and Slow Electron‐Detachment‐Dissociation/Electron‐Photodetachment‐Dissociation of a 21‐Mer Peptide

Electron detachment dissociation (EDD) and electron photodetachment dissociation (EPD) are relatively new dissociation methods that involve electron detachment followed by radical‐driven dissociation from multiply deprotonated species. EDD yields prompt dissociation whereas only electron detachment is obtained by EPD; subsequent vibrational activation of the charge‐reduced radical anion is required to obtain the product ions. Herein, the fragmentation patterns that were obtained by EDD and by vibrational activation of the charge‐reduced radical anions that were produced through EDD or EPD (activated‐EDD and activated‐EPD) were compared. The observed differences were related to the dissociation kinetics and/or the contribution of electron‐induced dissociation (EID). Time‐resolved double‐resonance experiments were performed to measure the dissociation rate constants of the EDD product ions. Differences in the formation kinetics were revealed between the classical EDD/EPD ′a^._i/′′x_j complementary ions and some ′a^._i/c_i/′′′z^._j product ions, which were produced with slower dissociation rate constants, owing to the presence of specific neighbouring side chains. A new fragmentation pathway is proposed for the formation of the slow‐kinetics ′a^._i ions.