Investigation of UV matrix-assisted laser desorption fourier transform mass spectrometry for peptides

Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Ultraviolet matrix-assisted laser desorption can be used to enhance formation of [M + H]⁺, [M + Na]⁺, and [M + K)⁺ ions from small peptides for Fourier transform mass spectrometry (FTMS). In accord with laser desorption (LD) time-of-flight experiments, matrices such as nicotinic acid and 2-pyrazinecarboxylic acid exhibit strong enhancement effects (i.e., formation of abundant protonated and cationized molecules for the analyte with virtually no fragment ions) for 266 nm LD/FTMS, whereas pyrazinedicarboxylic acid provides no matrix enhancement at this wavelength. Both sinapinic acid and coumarin-120 provide strong matrix enhancement effects for the 355-nm LD of peptides. For the small peptides examined in this study, no significant differences in the abundance of fragment ions were observed between the 266- and 355-nm wavelengths. Matrix-assisted LD/FTMS is useful for the generation and characterization of ions corresponding to protonated and cationized molecules from virtually all biological compounds with molecular weights up to 2000. The lack of observation of biological ions with m/ z > 2500 may be related to inefficient trapping of these laser-desorbed ions or instrumental detection limitations of FTMS and is under further investigation.     

Injection of reagent ions into the selvedge region in fast-atom bombardment mass spectrometry

A divided probe that incorporates a potassium aluminosilicate glass target and an analyte/glycerol matrix target, spatially separated, was used to inject potassium ions (K⁺) into the high-pressure “selvedge” region formed above the analyte/glycerol matrix target during fast-atom bombardment (FAB); [M+K]⁺ adduct ions that represent the types of gas-phase neutral molecules present in the selvedge region are observed. Computer modeling assisted in designing the divided target and an additional ion optical element for the FAB ion source to optimize interactions between K⁺ ions and the desorbed neutral molecules. The capability of injecting K⁺ ions into the FAB experiment has utility in both mechanistic studies and analyses. Experimental results here are consistent with a model for the desorption/ionization processes in FAB in which some types of neutral analyte molecules are desorbed intact and are subsequently protonated by glycerol chemical ionization. Unstable protonated molecules undergo unimolecular decomposition to yield observed fragment ions. The use of K⁺ cationization of analytes for molecular weight confirmation is demonstrated, as well as its utility in FAB experiments in which mixtures are encountered.     

Characteristic Fragmentation of Polysiloxane Monolayer Films by Bombardment with Monatomic and Polyatomic Primary Ions in TOF-SIMS

This study reports the characteristic fragmentation patterns from two polysiloxane polymers that form ordered overlayer on silver substrates. Results are compared for the bombardment of various monatomic and polyatomic projectiles of Cs⁺, C₆₀⁺ (10 keV), Bi₁⁺, and Bi₃⁺ (25 keV) in the high mass range time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra. Results are reported from sub-monolayer (solution cast) coverages of poly(dimethylsiloxane)s with the number average molecular weights (M_n) of 2200 and 6140 Da, respectively, and Langmuir-Blodgett monolayers of poly(methylphenylsiloxane) with molecular weights (MW) from 600 and 1000 Da. For each film, Bi projectiles resulted in the emission of positive silver cluster ions from the substrate under the polymer overlayer and peaks corresponding to silver cluster ions with larger mass were observed by impact of polyatomic 25 keV Bi₃⁺ projectiles. In addition, depending on the change of energy of Bi₃⁺, a different pattern of fragments was observed. With Cs⁺ and C₆₀⁺ impact, however, the emission of silver cluster ions was not detected. In the case of C₆₀⁺ impact for PDMS-6140, peaks corresponding to silver-cationized intact oligomers were not observed. In this paper, these results are explained by the possible bombardment mechanism for each projectile, based on its mass, energy, and split trajectories of the component atoms under the polyatomic impact.     

Peptide structural analysis using continuous Ar cluster and C60 ion beams

A novel application of time-of-flight secondary ion mass spectrometry (ToF-SIMS) with continuous Ar cluster beams to peptide analysis was investigated. In order to evaluate peptide structures, it is necessary to detect fragment ions related to multiple neighbouring amino acid residues. It is, however, difficult to detect these using conventional ToF-SIMS primary ion beams such as Bi cluster beams. Recently, C₆₀ and Ar cluster ion beams have been introduced to ToF-SIMS as primary ion beams and are expected to generate larger secondary ions than conventional ones. In this study, two sets of model peptides have been studied: (des-Tyr)-Leu-enkephalin and (des-Tyr)-Met-enkephalin (molecular weights are approximately 400 Da), and [Asn¹ Val⁵]-angiotensin II and [Val⁵]-angiotensin I (molecular weights are approximately 1,000 Da) in order to evaluate the usefulness of the large cluster ion beams for peptide structural analysis. As a result, by using the Ar cluster beams, peptide molecular ions and large fragment ions, which are not easily detected using conventional ToF-SIMS primary ion beams such as Bi₃ ⁺, are clearly detected. Since the large fragment ions indicating amino acid sequences of the peptides are detected by the large cluster beams, it is suggested that the Ar cluster and C₆₀ ion beams are useful for peptide structural analysis.     

Direct evidence for preformed ions of porphyrins in the solvent matrix for fast atom bombardment mass spectrometry

The observation that protonated molecules are present in solvents utilized for fast atom bombardment (FAB) mass spectrometric studies has been demonstrated using visible absorption spectrometry. Addition of porphyrins to thioglycerol, a solvent used for FAB analyses, results in partial protonation of the molecule. This reaction can be monitored by observing the shift in visible absorption maxima associated with the molecular transition from free base to protonated structure. A good correlation is observed between the degree of protonation indicated by the appropriate absorption bands and the abundance of the [M + H]⁺ ion in the FAB spectrum of the corresponding solution. Addition of certain non-polar porphyrin molecules to thioglycerol does not result in the protonation of the molecule in solution; in these cases, analyses of the corresponding solutions by FAB do not yield [M + H]⁺ ions. Subsequent addition of trifluoroacetic acid to the solvent has proved sufficient to protonate the analyte molecule, as indicated by the visible absorption spectrum; FAB analyses of these non-polar porphyins in acidified solvent result in the observation of [M + H]⁺ ions. These experiments demonstrate that analyses of these analyte molecules requires that they be present as ions in solution prior to analysis by FAB. This study provides experimental evidence for the presence of ions in solutions employed for FAB analysis, suggesting that these ions are essential for the generation of the protonated molecules observed during FAB mass spectrometric analyses.     

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.     

Organic cluster ions from continuous-flow fast atom bombardment

Cluster ions from fast atom bombardment of liquid alcohols and nitriles were examined using a continuous-flow technique. Protonated molecular M_nH⁺ species are the dominant cluster ions observed in molecules of formula M. The abundances of the M_nH⁺ cluster ions decrease monotonically with increasing n, and within a homologous series the M_nH⁺ abundance diminishes more rapidly for higher molecular mass compounds. Reaction products (ROH)_n(H₂O)H⁺ and (ROH)_n(ROR)H⁺ are observed also in the case of alcohols, and the ion abundances decrease with increasing n. Radiation damage yields fragment ions and ionic alkyl reaction products which are captured in solvent clusters. Semi-empirical molecular orbital methods were used to examine the energetics of cluster ion formation and decomposition pathways. Metastable decomposition processes exhibit only evaporative loss of monomers, with the probability of loss increasing sharply with n. The evaporative ensemble model of Klots was used to predict the cluster size-dependent trends of metastable dissociation processes observed for alcohol and nitrile cluster ions.     

Enhancing the ratio of molecular ions to non-covalent compounds in the electrospray interface of LC-MS in quantitative analysis

A common problem encountered during the development of MS methods for the quantitation of small organic molecules by LC-MS is the formation of non-covalently bound species or adducts in the electrospray interface. Often the population of the molecular ion is insignificant compared to those of all other forms of the analyte produced in the electrospray, making it difficult to obtain the sensitivity required for accurate quantitation. We have investigated the effects of the following variables: orifice potential, nebulizer gas flow, temperature, solvent composition and the sample pH on the relative distributions of ions of the types MH⁺, MNa⁺, MNH₄⁺, and 2MNa⁺, where M represents a small organic molecule: BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile). Orifice potential, solvent composition and the sample pH had the greatest influence on the relative distributions of these ions, making these parameters the most useful for optimizing methods for the quantitation of small molecules.     

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) of polyisoprenes

Polyisoprenes (PIPs) with average molecular weights from 650 to 800,000 Da have been studied by time-of-flight secondary ion mass Spectrometry (TOF-SIMS) in the static mode. Polymer samples were bombarded by argon primary ions, and positive SIMS spectra were collected. Effects of branching and unsaturation in the polymer structure on ion formation were studied. The pendant methyl group showed little tendency to fracture as a cation. In the low mass region, C_nH _2n–1 ⁺ appeared to be more intense than C_nH _2n+1 ⁺ , attributed to the double bond structure of polyisoprene. Additionally, ion formation varied as a function of polymer molecular weight. Cationized intact oligomers and fragments dominate the high mass region. Oligomer distributions were used to calculate average molecular weights for polyisoprenes. A statistical chain scission mechanism was used to qualitatively explain the formation of five clusters within a unique fragmentation pattern. Detailed studies of the cluster structure pointed out that each cluster contained several species having varied degrees of unsaturation. It is believed that double bond rearrangements occur.     

Fast atom bombardment mass spectra of telluronium salts

The fast atom bombardment (FAB) mass spectra of telluronium salts were studied. The spectra exhibit the intact cation (C⁺) and cluster ions ([M + C]⁺). The principal fragment ions in the FAB mass spectra of telluronium salts are [RTe]⁺, [R₂Te]⁺˙, [R₂Te − H]⁺, [RTeR′]⁺˙, and [RTeR′ + H]⁺. When the anion was [BPh₄]⁻, interesting cluster ions such as [M + C − BPh₃]⁺ appeared.     

Cationization of dendritic macromolecule adsorbates on metals studied by time‐of‐flight secondary ion mass spectrometry

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was utilized to study dendritic macromolecules with various architectures, such as dendrons, dendrimers and hyperbranched polyesters prepared from bis‐(hydroxymethyl)propionic acid (Bis‐MPA) and a series of hyperbranched polyethers based on 3‐ethyl‐3(hydroxymethyl)oxetane. The measurements were performed on spin‐coated thin films of the branched molecules (D) onto silicon, chemically etched copper foil and silver‐coated wafers. They showed weak signatures of molecular ions by proton capture (D + H)⁺ in the high mass range of the spectra (m/z > 400). On the contrary, cationization of the intact molecules with alkali or transition metal ions such as Na⁺, Cu⁺ or Ag⁺ was observed. High‐intensity quasi‐molecular ions (D + M)⁺ (with M = Na⁺, Cu⁺ or Ag⁺) allowed the studied polymers to be identified. The whole molecular species were observed for Bis‐MPA dendrons and dendrimers up to 3000 Da for hydroxyl or acetonide‐terminated derivatives. The success of the so‐called cationization experiments with metal substrates compared with analysis of molecular adsorbates on silicon is highlighted. The ToF‐SIMS sensitivity appeared useful to provide information about the molecular end‐groups or to highlight incomplete reaction occurring during some deprotection step of the synthesis. Only uncationized fragments of low masses were detected for the hyperbranched polyesters. This result suggested the effect of molecular asymmetry and/or flattening of the molecules on the substrates, which hampered the molecule lift‐off efficiency. Nevertheless, the hyperbranched polyethers were characterized based on the peak distribution of intensities, which allowed estimation of their molecular weight average. This work was intended to illustrate the capabilities of ToF‐SIMS to analyse dendritic polymers on surfaces. Copyright © 2003 John Wiley & Sons, Ltd.     

Improvement of biological time-of-flight-secondary ion mass spectrometry imaging with a bismuth cluster ion source

A new liquid metal ion gun (LMIG) filled with bismuth has been fitted to a time-of-flight-secondary ion mass spectrometer (TOF-SIMS). This source provides beams of Bi(n)q+ clusters with n = 1-7 and q = 1 and 2. The appropriate clusters have much better intensities and efficiencies than the Au3+ gold clusters recently used in TOF-SIMS imaging, and allow better lateral and mass resolution. The different beams delivered by this ion source have been tested for biological imaging of rat brain sections. The results show a great improvement of the imaging capabilities in terms of accessible mass range and useful lateral resolution. Secondary ion yields Y, disappearance cross sections sigma, efficiencies E = Y/sigma , and useful lateral resolutions deltaL have been compared using the different bismuth clusters, directly onto the surface of rat brain sections and for several positive and negative secondary ions with m/z ranging from 23 up to more than 750. The efficiency and the imaging capabilities of the different primary ions are compared by taking into account the primary ion current for reasonable acquisition times. The two best primary ions are Bi3+ and Bi5(2+). The Bi3+ ion beam has a current at least five times larger than Au3+ and therefore is an excellent beam for large-area imaging. Bi5(2+) ions exhibit large secondary ions yields and a reasonable intensity making them suitable for small-area images with an excellent sensitivity and a possible useful lateral resolution <400 nm.     

Chemical ionization mass spectrometry of halogenoalkanes with tetramethylsilane as reagent gas

Chemical ionization mass spectra of halogenoalkanes (RX) obtained using tetramethylsilane as reagent gas show two major peaks corresponding to the cluster ion RX⁺SiMe₃ and alkyl ion R⁺. Iodides exhibit the highest affinity toward the trimethylsilyl ion and produce the most stable silylated molecular ions, whereas bromides and especially chlorides are less reactive toward Me₃Si⁺ ions and form less stable [M + SiMe₃]⁺ ions.     

Behaviour of methyl red under fast atom bombardment conditions

The influence of the pH, the nature of the matrix and the presence of a surfactant on the positive- and negative-ion abundances in the molecular mass region in the fast atom bombardment (FAB) mass spectra of methyl red was investigated. A small but significant pH effect was observed which was attributed to the non- or at the most low surface-active character of the intact methyl red molecule. As expected, the more basic the solution, the less protonated molecules with respect to M⁺˙ are observed and in the negative-ion mode less [M + H]^? and more [M – H]^? ions with respect to M^?˙ were found. In contrast to neutral solutions, both acidic and basic solutions give a long-lasting stable response of all methyl red ions. For dyes with a moderately negative redox potential such as methyl red, beam-induced redox reactions seem to play a role in the ionization process, the neutral medium offering the best conditions for reduction processes. The ion intensities in the molecular mass region depend on the nature of the matrix. Protonation of the molecule has been found to be more effective in glycerol than in 3-nitrobenzyl alcohol; the former also appears to offer the best conditions for reduction processes. Anionic and cationic surfactants effectively suppress the contribution of ions from glycerol in both positive- and negative-ion spectra and generally promote the formation of analyte ions at the surface. The most important effect of the surfactant in a neutral medium seems to be the promotion of a regular transport of ions and molecules to the surface, which permits the creation of stable ion currents, instead of an unstable ion beam if the surfactant is absent. Moreover, when the surfactant is present an increase of the sample ion abundances is observed. Redox reactions involving molecules and molecular ions and also molecules and preformed ions in the solution, brought to the surface by micelles, have been proposed to give some contribution for the small but significant enhanced abundance of [M + nH]⁺ (n > 1) ions with respect to [M + H]⁺ ions, in the presence of a surfactant. The results have been rationalized in terms of the surface phenomena while the important role of surfactants for obtaining better FAB mass spectra is emphasized.     

Introduction to time-of-flight secondary ion mass spectrometry application in chromatographic analysis

New on-line analytical system coupling thin layer chromatography (TLC) and high selective identification unit-time of flight secondary ion mass spectrometry (TOF-SIMS) is introduced in this article. Chromatographic mixture separation and analyte surface deposition followed with surface TOF-SIMS analysis on-line allows to identify the analytes at trace and ultratrace levels. The selected analytes with different detectability and identification possibility were analysed in this hyphenated unit (Methyl Red indicator, Terpinolen and Giberrelic acid). Here, the chromatographic thin layer plays a universal role: separation unit, analyte depositing surface and TOF-SIMS interface, finally. Two depositing substrates and TOF-SIMS compatible interfaces were tested in above-mentioned interfacing unit: modified aluminium backed chromatographic thin layer and monolithic silica thin layer. The sets of positive and negative ions TOF-SIMS spectra obtained from different SIMS modes of analysis were used for analyte identification purposes. SIMS enables analyte detection with high mass resolution at the concentration level that is not achieved by other methods.     

RIMS analysis of ion induced fragmentation of molecules sputtered from an enriched U3O8 matrix

Resonance ionization mass spectrometry was used to measure the composition of the sputtered flux from 15 keV Ga⁺, Au⁺, Au₂ ⁺ and Au₃ ⁺ primary ions impacting a ²³⁵U enriched U₃O₈ standard. We demonstrate that molecular fragmentation decreases as the primary ion mass and nuclearity increases. Stopping and range of ions in matter calculations show that cluster ions (Au₂ ⁺ and Au₃ ⁺) deposit more of their energy via direct knock-ons with near-surface target atoms, whereas monatomic ions (Ga⁺ and Au⁺) penetrate much deeper into the target sub-surface region. We correlate these results to the experimental observations by showing that increased cluster ion sputter yields partition the projectile energy over a larger number of sputtered molecules. Therefore, while cluster ions deposit more total energy into the near surface region of the target compared to monatomic ions, the energy per molecule decreases with projectile mass and nuclearity. Less energy per molecule decreases the number of U–O bond breaks and, consequently, leads to a decrease in molecular fragmentation. Additionally, the extent of molecular fragmentation as a function of ion dose was evaluated. We show that molecular fragmentation increases with increased ion dose; primarily as a result of sub-surface chemical damage accumulation. The relative intensity of this effect appears to be projectile independent.     

Study of adsorption of tetraethylammonium ions on Bi single crystal planes from solutions in ethanol

The adsorption of tetraethylammonium (TEA⁺) ions on the (001) and planes of the bismuth single crystal from solutions in ethanol has been investigated by impedance measurement method. The experimental data were obtained in 0.02 M mixed-electrolyte solutions and the calculations performed with electrode potential as the independent electrical variable. The Gibbs energy of adsorption of TEA⁺ ions has been calculated using the simple virial adsorption isotherm, and it was found that the adsorption of TEA⁺ cations is weaker than the adsorption of halide anions. The electrosorption valency evaluated has a nearly constant value in the potential region studied. It was concluded that the formed effective surface dipole is significantly screened by the solvent molecules and the metal electron gas. The analysis of the impedance spectra was performed by fitting the experimental data to the various equivalent circuits. It was found that the behaviour of TEA⁺ ions at Bi(hkl)∣ethanol interface can be described with the equivalent circuit, corresponding to the classical Frumkin–Melik-Gaikazyan model. The results obtained indicate that only weak interaction between TEA⁺ ions and bismuth surface takes place, and there is no remarkable partial charge transfer from the adsorbed ions to the Bi surface atoms.     

Thermal surface ionization of some complex salts

Thermal ionization on the surface of a heated wire is applied to the volatilization products of alkali salts of carboxylic and sulfonic acids, and to quaternary ammonium salts. The mass spectra of the alkali salts exhibit almost exclusively cationized molecules. They provide evidence for the evaporation of intact clustered molecules even under conditions of a slow rate of evaporation of the salts. The method appears to be of interest for selective detection of alkali salts from complex mixtures. With the ammonium salts [R₄N]⁺ ions are formed by thermal surface ionization of intact salt molecules. The evaporation of these molecules could be detected at rather low temperatures.     

Reagent Cluster Anions for Multiple Gas-Phase Covalent Modifications of Peptide and Protein Cations

Multiple gas phase ion/ion covalent modifications of peptide and protein ions are demonstrated using cluster-type reagent anions of N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate) and 2-formyl-benzenesulfonic acid (FBMSA). These reagents are used to selectively modify unprotonated primary amine functionalities of peptides and proteins. Multiple reactive reagent molecules can be present in a single cluster ion, which allows for multiple covalent modifications to be achieved in a single ion/ion encounter and at the ‘cost’ of only a single analyte charge. Multiple derivatizations are demonstrated when the number of available reactive sites on the analyte cation exceeds the number of reagent molecules in the anionic cluster (e.g., data shown here for reactions between the polypeptide [K₁₀ + 3H]³⁺ and the reagent cluster [5R_5Na – Na]^–). This type of gas-phase ion chemistry is also applicable to whole protein ions. Here, ubiquitin was successfully modified using an FBMSA cluster anion which, upon collisional activation, produced fragment ions with various numbers of modifications. Data for the pentamer cluster are included as illustrative of the results obtained for the clusters comprised of two to six reagent molecules.

Study of adduct ions of meso-phenyl-substituted tetrabenzoporphyrins by fast-atom bombardment mass spectrometry

Mass spectra of meso-phenyl-substituted tetrabenzoporphyrins were investigated by fast-atom bombardment mass spectrometry and tandem mass spectrometry. A cluster of adduct ions with mass-to-charge ratio values higher than the corresponding molecular ions of the porphyrins has been observed. The mass number differences among the series of cluster ions are constant depending on the para-phenyl substituents. Under certain conditions, dimers or trimers of molecular ions with low abundances have been detected. To trace the origin of the adduct ions, a series of experiments based on mass spectrometry have been carried out. The mass spectrum of tetrabenzoporphyrin showed no adduct ions with mass number differences of 90 even with the addition of phenylacetic acid. The mass spectrum of meso-tetraphenylte-trabenzoporphyrin ¹³C-labeled at the meso carbons showed adduct ions with mass number differences of 91. Product spectra of [2M + H]⁺ or [3M + H]⁺ of porphyrins exhibited adduct ions. All these results suggest that fragmentations of [2M + H]⁺ or [3M + H]⁺ may be one of the many possible routes to form the adduct ions, and the mass number differences among the series of these cluster ions should correspond to the benzyl group from the meso positions of meso-phenyl-substituted tetrabenzoporphyrins.