Surface-induced dissociation of ions produced by matrix-assisted laser desorption/ionization in a fourier transform ion cyclotron resonance mass spectrometer

Intermediate pressure matrix-assisted laser desorption/ionization (MALDI) source was constructed and interfaced with a 6-T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially configured for surface-induced dissociation (SID) studies. First MALDI-SID results in FT-ICR are presented, demonstrating unique advantages of SID over conventional FT-ICR MS ion activation techniques for structural characterization of singly protonated peptide ions. Specifically, we demonstrate that SID on a diamond surface results in a significantly better sequence coverage for singly protonated peptides than SORI-CID. A combination of two effects contributes to the improved sequence coverage: shattering of peptide ions on surfaces opens up a variety of dissociation channels at collision energies above 40 eV, and second, wide internal energy distribution deposited by collision with a stiff diamond surface provides an efficient mixing between the primary reaction channels that are dominant at low internal energies and extensive fragmentation at high internal excitation that results from shattering. Activation of MALDI-generated ions by collisions with surfaces in FT-ICR MS is a new powerful method for characterization and identification of biomolecules     

Surface modification and patterning using low-energy ion beams: Si-O bond formation at the vacuum/adsorbate interface.

Modification of hydroxyl-terminated self-assembled monolayer (HO-SAM) surfaces by collision of low-energy (15 eV) hyperthermal Si(CH3)3+ ions is shown to lead to Si-O bond formation and terminal trimethylsilyl ether formation. Modification was verified by in situ mass spectrometry using chemical sputtering with CF3+ ions (70 eV), ex situ secondary ion mass spectrometric analysis (12 kV Ga+ primary ion beam), and through X-ray photoelectron spectroscopy by monitoring Si (2s). The nature of the surface modification was further established by analysis of synthetic SAM surfaces made up of mixtures of the trimethylsilyl-11-mercapto-1-undecane ether and various proportions of the hydroxyl-terminated mercaptan (11-mercapto-1-undecanol). These mixed surfaces, as well as the spectroscopic data, indicate that ca. 30% of the hydroxyl chains are covalently modified at saturation coverage. Analogous surface transformations are achieved using Si(CH3)2F+ and Si(CH3)2C6H5+.     

Ambient ion soft landing

Ambient ion soft landing, a process in which polyatomic ions are deposited from air onto a surface at a specified location under atmospheric pressure, is described. Ions generated by electrospray ionization are passed pneumatically through a heated metal drying tube, their ion polarity is selected using ion deflectors, and the dry selected ions are soft-landed onto a selected surface. Unlike the corresponding vacuum soft-landing experiment, where ions are mass-selected and soft-landed within a mass spectrometer, here the ions to be deposited are selected through the choice of a compound that gives predominantly one ionic species upon ambient ionization; no mass analysis is performed during the soft landing experiment. The desired dry ions, after electrical separation from neutrals and counterions, are deposited on a surface. Characterization of the landed material was achieved by dissolution and analysis using mass spectrometry or spectrofluorimetry. The treated surface was also characterized using fluorescence microscopy, which allowed surfaces patterned with fluorescent compounds to be imaged. The pure dry ions were used as reagents in heterogeneous ion/surface reactions including the reaction of pyrylium cations with d-lysine to form the N-substituted pyridinium cation. The charged microdroplets associated with incompletely dried ions could be selected for soft landing or surface reaction by choice of the temperature of a drying tube inserted between the ion source and the electrical ion deflectors.     

Design and performance of an instrument for soft landing of biomolecular ions on surfaces

A new ion deposition apparatus was designed and constructed in our laboratory. Our research objectives were to investigate interactions of biomolecules with hydrophilic and hydrophobic surfaces and to carry out exploratory experiments aimed at highly selective deposition of spatially defined and uniquely selected biological molecules on surfaces. The apparatus includes a high-transmission electrospray ion source, a quadrupole mass filter, a bending quadrupole that deflects the ion beam and prevents neutral molecules originating in the ion source from impacting the surface, an ultrahigh vacuum (UHV) chamber for ion deposition by soft landing, and a vacuum lock system for introducing surfaces into the UHV chamber without breaking vacuum. Ex situ analysis of surfaces following soft landing of mass-selected peptide ions was performed using 15 keV Ga+ time-of-flight secondary ion mass spectrometry and grazing incidence infrared reflection-absorption spectroscopy. It is shown that these two techniques are highly complementary methods for characterization of surfaces prepared with a range of doses of mass-selected biomolecular ions. We also demonstrated that soft landing of peptide ions on surfaces can be utilized for controlled preparation of peptide films of known coverage for fundamental studies of matrix effects in SIMS.     

Vibrational spectroscopy and mass spectrometry for characterization of soft landed polyatomic molecules

We report implementation of two powerful characterization tools, in situ secondary ion mass spectrometry (SIMS) and ex situ surface enhanced Raman spectroscopy (SERS), in analyzing surfaces modified by ion soft landing (SL). Cations derived from Rhodamine 6G are soft landed onto Raman-active silver colloidal substrates and detected using SERS. Alternatively and more conveniently, high-quality SERS data are obtained by spin coating a silver colloidal solution over the modified surface once SL is complete. Well-defined SERS features are observed for Rhodamine 6G in as little as 15 min of ion deposition. Deposition of ~3 pmo1 gave high-quality SERS spectra with characteristic spectroscopic responses being derived from just ~0.5 fmol of material. Confocal SERS imaging allowed the enhancement to be followed in different parts of deposited dried droplets on surfaces. Characteristic changes in Raman spectral features occur when Rhodamine 6G is deposited under conditions that favor gas-phase ion fragmentation. Simultaneous deposition of both the intact dye and its fragment ion occurs and is confirmed by SIMS analysis. The study was extended to other Raman active surfaces, including Au nanostar and Au coated Ni nanocarpet surfaces and to SL of other molecules including fluorescein and methyl red. Overall, the results suggest that combination of SERS and SIMS measurements are effective in the characterization of surfaces produced by ion SL with significantly enhanced molecular specificity.     

Analysis of VX on soil particles using ion trap secondary ion mass spectrometry.

The direct detection of the nerve agent VX (methylphosphonothioic acid, S-[2-[bis(1-methylethyl)amino]ethyl] O-ethyl ester) on milligram quantities of soil particles has been achieved using ion trap secondary ion mass spectrometry (IT-SIMS). VX is highly adsorptive toward a wide variety of surfaces; this attribute makes detection using gas-phase approaches difficult but renders the compound very amenable to surface detection. An ion trap mass spectrometer, modified to perform SIMS, was employed in the present study. A primary ion beam (ReO4-) was fired on axis through the ion trap, where it impacted the soil particle samples. [VX + H]+, [VX + H]+ fragment ions, and ions from the chemical background were sputtered into the gas-phase environment of the ion trap, where they were either scanned out or isolated and fragmented (MS2). At a surface concentration of 0.4 monolayer, intact [VX + H]+, and its fragment ions, were readily observable above background. However, at lower concentrations, the secondary ion signal from VX became obscured by ions derived from the chemical background on the surface of the soil particles. MS2 analysis using the ion trap was employed to improve detection of lower concentrations of VX: detection of the 34S isotopic ion of [VX + H]+, present at a surface concentration of approximately 0.002 monolayer, was accomplished. The study afforded the opportunity to investigate the fragmentation chemistry of VX. Semiempirical calculations suggest strongly that the molecule is protonated at the N atom. Deuterium labeling showed that formation of the base peak ion (C2H4)N(i-C3H7)2+ involves transfer of the amino proton to the phosphonothioate moiety prior to, or concurrent with, C-S bond cleavage. To manage the risk associated with working with the compound, the vacuum unit of the IT-SIMS was located in a hood, connected by cables to the externally located electronics and computer.     

Carbon-13 labeled polymers: an alternative tracer for depth profiling of polymer films and multilayers using secondary ion mass spectrometry

13C labeling is introduced as a tracer for depth profiling of polymer films and multilayers using secondary ion mass spectrometry (SIMS). Deuterium substitution has traditionally been used in depth profiling of polymers but can affect the phase behavior of the polymer constituents with reported changes in both bulk-phase behavior and surface and interfacial interactions. SIMS can provide contrast by examining various functional groups, chemical moieties, or isotopic labels. 13C-Labeled PS (13C-PS) and unlabeled PS (12C-PS) and PMMA were synthesized using atom-transfer radical polymerization and assembled in several model thin-film systems. Depth profiles were recorded using a Cameca IMS-6f magnetic sector mass spectrometer using both 6.0-keV impact energy Cs+ and 5.5-keV impact energy O2+ primary ion bombardment with detection of negative and positive secondary ions, respectively. Although complete separation of 12C1H from 13C is achieved using both primary ion species, 6.0-keV Cs+ clearly shows improved detection sensitivity and signal-to-noise ratio for detection of 12C, 12C1H, and 13C secondary ions. The use of Cs+ primary ion bombardment results in somewhat anomalous, nonmonotonic changes in the 12C, 12C1H, and 13C secondary ion yields through the PS/PMMA interface; however, it is shown that this behavior is not due to sample charging. Through normalization of the 13C secondary ion yield to the total C (12C + 13C) ion yield, the observed effects through the PS/PMMA interface can be greatly minimized, thereby significantly improving analysis of polymer films and multilayers using SIMS. Mass spectra of 13C-PS and 12C-PS were also analyzed using a PHI TRIFT I time-of-flight mass spectrometer, with 15-keV Ga+ primary ion bombardment and detection of positive secondary ions. The (12)C7(1)H7 ion fragment and its 13C-enriched analogues have significant secondary ion yields with negligible mass interferences, providing an early indication of the potential for future use of this technique for cluster probe depth profiling of high molecular weight 13C-labeled fragments.     

Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors

Electron-transfer dissociation (ETD) delivers the unique attributes of electron capture dissociation to mass spectrometers that utilize radio frequency trapping-type devices (e.g., quadrupole ion traps). The method has generated significant interest because of its compatibility with chromatography and its ability to: (1) preserve traditionally labile post-translational modifications (PTMs) and (2) randomly cleave the backbone bonds of highly charged peptide and protein precursor ions. ETD, however, has shown limited applicability to doubly protonated peptide precursors, [M + 2H]2+, the charge and type of peptide most frequently encountered in "bottom-up" proteomics. Here we describe a supplemental collisional activation (CAD) method that targets the nondissociated (intact) electron-transfer (ET) product species ([M + 2H]+*) to improve ETD efficiency for doubly protonated peptides (ETcaD). A systematic study of supplementary activation conditions revealed that low-energy CAD of the ET product population leads to the near-exclusive generation of c- and z-type fragment ions with relatively high efficiency (77 +/- 8%). Compared to those formed directly via ETD, the fragment ions were found to comprise increased relative amounts of the odd-electron c-type ions (c+*) and the even-electron z-type ions (z+). A large-scale analysis of 755 doubly charged tryptic peptides was conducted to compare the method (ETcaD) to ion trap CAD and ETD. ETcaD produced a median sequence coverage of 89%-a significant improvement over ETD (63%) and ion trap CAD (77%).     

SIMS of organic anions adsorbed onto an aminoethanethiol self-assembled monolayer: an approach for enhanced secondary ion emission

Secondary ion mass spectrometry (SIMS) was used to monitor the uptake of organic anions from solution by aminoethanethiol (AET) monolayers on Au substrates, as a test of the applicability of this monolayer as a substrate for organic SIMS analysis. Event-by-event bombardment and detection mode coupled with coincidence counting allowed the atomic and polyatomic projectile impacts on a particular sample surface to be compared simultaneously and under the same experimental conditions. The mass spectra produced from the monolayer surface and those from Au and Si blanks demonstrate that the AET monolayer is important to the uptake of the organic anion. The exchanged monolayer surfaces were used to measure secondary ion yields, defined as the number of secondary ions detected per incident primary ion, produced from ultrathin films by (CsI)nCs+ (n = 0-2) projectiles at the limit of single-ion impacts. The yield of a tetradecyl sulfate (IDS) anion was improved by a factor of 200 using the AET substrate instead of the thick salt target. The intact ion and fragment ion yield trends produced from the AET surface were measured as a function of number of atoms in the primary projectile and energy. We observed a yield increase for both the intact ion and the fragment ion with the projectile complexity and energy. The increase in yield per projectile atom was linear for the emission of intact TDS and intact dodecyl sulfate from the AET surfaces. A supralinear yield enhancement, however, was observed for the fragment ion SO3- when the three-atom (CsI)Cs+ cluster was used. The experiments demonstrate that the various organosulfate and suffonates are weakly bound to the AET surface and their adsorption to the AET monolayer is reversible. The utility of the AET monolayer on Au was also tested as a general substrate for the characterization of derivatized organic molecules with biological and industrial importance by TOF-SIMS.     

Quantitative SERS Measurements on Dielectric-Overcoated Silver-Island Films by Solution-Deposition Control of Surface Concentrations

A simple method to control the dosing of small adsorbate molecules onto solid surfaces from liquid solution is applied to quantitative surface-enhanced Raman scattering measurements on dielectric-overcoated silver-island films. The deposition method, based on substrate withdrawal from solution, is evaluated by measuring fluorescence (ex situ) and optical absorption (in situ) of dye molecules deposited onto glass surfaces. Control of adsorbate surface concentrations was accomplished by varying the withdrawal rate and the concentration of the dye in solution. The dosing method was used to study the dependence of the electromagnetic contribution to SERS enhancement on surface coverage of scatterer. The sensitivity enhancement was found to be constant for adsorbate coverages up to 60-80% of a monolayer. Beyond a full monolayer, SERS enhancement for additional molecules deposited onto the surface was found to drop significantly, by as much as 1 order of magnitude.     

Electron transfer ion/ion reactions in a three-dimensional quadrupole ion trap: reactions of doubly and triply protonated peptides with SO2*-

Ion-ion reactions between a variety of peptide cations (doubly and triply charged) and SO2 anions have been studied in a 3-D quadrupole ion trap, resulting in proton and electron transfer. Electron transfer dissociation (ETD) gives many c- and z-type fragments, resulting in extensive sequence coverage in the case of triply protonated peptides with SO2*-. For triply charged neurotensin, in which a direct comparison can be made between 3-D and linear ion trap results, abundances of ETD fragments relative to one another appear to be similar. Reactions of doubly protonated peptides with SO2*- give much less structural information from ETD than triply protonated peptides. Collision-induced dissociation (CID) of singly charged ions formed in reactions with SO2*- shows a combination of proton and electron transfer products. CID of the singly charged species gives more structural information than ETD of the doubly protonated peptide, but not as much information as ETD of the triply protonated peptide.     

Protocols for three-dimensional molecular imaging using mass spectrometry

A protocol for three-dimensional molecular thin-film analysis is described that utilizes imaging time-of-flight secondary ion mass spectrometry and large-area atomic force microscopy. As a test study, a 300-nm trehalose film deposited on a Si substrate was structured by bombardment with a focused 15-keV Ga+ ion beam and analyzed using a 40-keV C60+ cluster ion beam. A three-dimensional sputter depth profile was acquired as a series of high-resolution lateral SIMS images with intermittent erosion cycles. As the most important result of this study, we find that the structured film exhibits a highly nonuniform erosion rate, thus preventing a simple conversion of primary ion fluence into eroded depth. Instead, the depth scale calibration must be performed individually on each pixel of the imaged area. The resulting laterally resolved depth profiles are discussed in terms of the chemical damage induced by the Ga+ bombardment along with the physics of the C60+ induced erosion process.     

Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry

One of the major factors governing the "top-down" sequence analysis of intact multiply protonated proteins by tandem mass spectrometry is the effect of the precursor ion charge state on the formation of product ions. To more fully understand this effect, electrospray ionization coupled to a quadrupole ion trap mass spectrometer, collision-induced dissociation, and gas-phase ion/ion reactions have been employed to examine the fragmentation of the [M + 12H]12+ to [M + H]+ ions of bovine ubiquitin. At low charge states (+1 to +6), loss of NH3 or H2O from the protonated precursor and directed cleavage at aspartic acid residues was observed. At intermediate charge states, (+7, +8, and +9), extensive nonspecific fragmentation of the protein backbone was observed, with 50% sequence coverage obtained from the [M + 8H]8+ ion alone. At high charge states, (+10, +11, +12), the single dominant channel that was observed was the preferential fragmentation of a single proline residue. These data can be readily explained in terms of the current model for intramolecular proton mobilization, that is, the "mobile proton model", the mechanisms for amide bond dissociation developed for protonated peptides, as well as the structures of the multiply charged ions of ubiquitin in the gas phase, examined by ion mobility and hydrogen/deuterium exchange measurements.     

Preparative soft and reactive landing of multiply charged protein ions on a plasma-treated metal surface

Soft landing on a plasma-treated metal surface of multiply protonated protein ions from the gas phase results in a substantial retention of protein function, as demonstrated for trypsin and streptavidin. The majority of trypsin ions soft-landed at hyperthermal kinetic energies are undamaged and retain 72-98% of enzymatic activity after being washed into solution. A small fraction of trypsin ions that were landed at nominal kinetic energies of 130-200 eV remain tethered to the surface and show approximately 50% enzymatic activity. The streptavidin tetramer is found to dissociate to monomer units upon multiple charging in electrospray. The majority of soft-landed monomers can be washed into solution where they show affinity to biotin. The layer of streptavidin monomer that is immobilized on the surface can be detected if fluorescence-tagged and retains the ability to reversibly bind biotin. A mechanism is proposed to explain nondestructive protein ion discharge on the surface that considers proton migration from the soft-landed cations to the metal oxide layer and metal ion reduction by electron transfer from the bulk metal.     

Two-dimensional ECD FT-ICR mass spectrometry of peptides and glycopeptides

2D FT-ICR MS allows the correlation between precursor and fragment ions by modulating ion cyclotron radii for fragmentation modes with radius-dependent efficiency in the ICR cell without the need for prior ion isolation. This technique has been successfully applied to ion-molecule reactions, Collision-induced dissociation and infrared multiphoton dissociation. In this study, we used electron capture dissociation for 2D FT-ICR MS for the first time, and we recorded two-dimensional mass spectra of peptides and a mixture of glycopeptides that showed fragments that are characteristic of ECD for each of the precursor ions in the sample. We compare the sequence coverage obtained with 2D ECD FT-ICR MS with the sequence coverage obtained with ECD MS/MS and compare the sensitivities of both techniques. We demonstrate how 2D ECD FT-ICR MS can be implemented to identify peptides and glycopeptides for proteomics analysis.     

Cyclophosphoramidate ion as mass defect marker for efficient detection of protein serine phosphorylation

A novel method is reported to modify the phosphate groups on phosphoserine peptides to the corresponding phosphoramidates, using 2-aminobenzylamine. Upon collision-induced dissociation, the modified peptides release the positively charged phosphoramidate that via gas-phase intramolecular elimination forms a cyclophosphoramidate (CyPAA) ion, the protonated form of 1,4-dihydro-2-hydroxy-2-oxobenzo[3,1,2]oxazaphosphorine. The positive nature of the ion eliminates the need for real-time instrument polarity switching and greatly increases the versatility of commonly used mass spectrometers for phosphopeptide analysis. This ion has sufficient mass defect, due to containing a phosphorus atom and a high content of oxygen atoms, which makes mass spectrometers of medium mass resolution and accuracy adequate for separating the ion from isobaric interfering ions. The specificity of the CyPAA ion for detecting phosphoserine peptides in complex peptide mixtures is comparable to the specificity of the phosphotyrosine immonium ion for phosphotyrosine peptides, allowing the efficient data complexity reduction for fast and focused analysis of phosphoserine-containing peptides.     

Alternately pulsed nanoelectrospray ionization/atmospheric pressure chemical ionization for ion/ion reactions in an electrodynamic ion trap

The alternate operation of nanoelectrospray ionization and atmospheric pressure chemical ionization, using a common atmosphere/vacuum interface and ion path, has been implemented to facilitate ion/ion reaction experiments in a linear ion trap-based tandem mass spectrometer. The ion sources are operated in opposite polarity modes whereby one of the ion sources is used to form analyte ions while the other is used to form reagent ions of opposite polarity. This combination of ion sources is well-suited to implementation of experiments involving multiply charged ions in reaction with singly charged ions of opposite polarity. Three analytically useful ion/ion reaction types are illustrated: the partial deprotonation of a multiply protonated protein, the partial protonation of a multiply deprotonated oligonucleotide, and electron transfer to a multiply protonated peptide. The approach described herein is attractive in that it enables both single proton-transfer and single electron-transfer ion/ion reaction experiments to be implemented without requiring major modifications to the tandem mass spectrometer hardware. Furthermore, a wide range of reactant ions can be formed with these ionization methods and the pulsed nature of operation appears to lead to no significant compromise in the performance of either ion source.     

Surface plasmon resonance analysis of Alzheimer's beta-amyloid aggregation on a solid surface: from monomers to fully-grown fibrils

We analyzed the aggregation of Alzheimer's beta-amyloid (1-42) (Abeta42) peptides from fresh monomers to fully grown fibrils by using in situ surface plasmon resonance (SPR) spectrometry and ex situ atomic force microscopy (AFM). To immobilize Abeta42 peptide on an SPR chip surface, different carboxy-terminated surfaces were investigated: (1) self-assembled monolayer of 11-mercaptoundecanoic acid and (2) carboxylated dextran-modified surface. It was found that the carboxylated dextran surface was more appropriate due to a much lower degree of nonspecific binding. By using the carboxylated dextran surface, we further investigated effects of key environmental factors, such as the density of surface-bound Abeta42, the concentration of Abeta42 in solution phase, and the presence of Fe3+ ions on Abeta42 fibrillation. The increase in either the surface density of Abeta42 or its concentration in incubation solution highly accelerated the formation of amyloid fibrils on the chip surface. The presence of Fe3+ ions in the incubation solution induced significantly denser aggregates, resulting in a nearly 6-fold increase of SPR angle shift. This work shows that SPR analysis coupled with AFM can be effectively used for analyzing amyloid aggregation and deposition on a solid surface from the very beginning to fully grown fibrils.     

High-resolution TOF-SIMS study of varying chain length self-assembled monolayer surfaces

A high-resolution time-of-flight secondary ionization mass spectrometer (TOF-SIMS) has been used to investigate chain length effects in hydrocarbon seff-assembled monolayer (SAM) surfaces on gold substrates. A wide range of n-alkanethiols was used to make homogeneous SAM surfaces, which included both odd and even hydrocarbon chain length thiols. Variations in coverage, extent of oxidation, and high-mass cluster formation as a function of hydrocarbon chain length of the alkanethiol SAM surfaces were investigated. Long-short chain length effects were observed for the relative coverage of the SAM surfaces, which directly influences the extent of oxidation for the thin films. The formation of gold-sulfur and gold-adsorbate cluster ions was also observed, since the mass range of the TOF-SIMS made it possible to monitor all of the cluster ions that were formed following the high-energy ion/surface interactions.     

Surface modification using a commercial triple quadrupole mass spectrometer

We report instrumental modifications to a commercial mass spectrometer that allow surface modification experiments to be performed using low-energy (electronvolt range) mass-selected ion beams. The design of the detector housing allows placement of the surface on the ion optical axis and some distance beyond the off-axis detector. Manipulation of the potentials applied to the final lens, detector housing, conversion dynode, and electron multiplier allow the ions to pass through the detector housing and impinge upon the surface without loss of the normal mode of detector operation. Ex situ analysis of the modified surface is performed using a home-built multisector mass spectrometer. The ability to modify organic thin films is demonstrated by a number of soft landing and surface modification experiments including (i) soft landing of (CH3)2SiNCS+ ions formed from trimethylsilyl isothiocyanate upon a fluorinated self-assembled monolayer (F-SAM) surface, (ii) soft landing and dissociative soft landing of the pseudomolecular cation of triphenylpyrylium tetrafluoroborate, viz. the triphenylpyrylium cation, upon an F-SAM surface, (iii) dissociative soft landing of 35ClCH2(CH3)2SiOSi(CH3)2+ formed from 1,3-bis(chloromethyl)disiloxane upon an F-SAM surface, (iv) surface passivation by reaction of the trimethylsilyl cation, Si(CH3)3+, with a hydroxyl-terminated self-assembled monolayer (OH-SAM), and (v) transhalogenation by reaction of CCl3+ (m/z 119) with an F-SAM surface.