Surface mass spectrometry of biotinylated self-assembled monolayers

Biotin and biotinylated self-assembled monolayers (SAMs) on gold have been investigated using time-of-flight secondary ion mass spectrometry, direct laser desorption, laser desorption with 193 nm photoionization of ion- and laser-desorbed species, and laser desorption with vacuum ultraviolet (VUV, 118 nm) photoionization. Our results indicate that direct laser desorption and laser desorption combined with 193 nm multiphoton ionization can detect a chromophoric molecule like biotin that is covalently bound to a SAM. However, secondary ion mass spectra were dominated by fragmentation, and ion desorption/193 nm photoionization detected no species related to biotin. The dominant features of the laser desorption/VUV mass spectra were neat and Au-complexed dimers of intact and fragmented biotinylated SAM molecules. Multiphoton and single-photon ionization of laser-desorbed neutrals from biotinylated SAMs both led to the production of ions useful for chemical analysis of the monolayer. Multiphoton ionization with ultraviolet radiation was experimentally less challenging but required a chromophore for ionization and resulted in significant fragmentation of the adsorbate. Single-photon ionization with VUV radiation was experimentally more challenging but did not require a chromophore and led to less fragmentation. X-ray photoelectron spectra indicated that the biotinylated SAM formed a disordered, 40-60 ? thick monolayer on Au. Additionally, projection photolithography with a Schwarzschild microscope was used to pattern the biotinylated SAM surface and laser desorption/photoionization was used to detect biotinylated adsorbates from the ～10 μm sized pattern.     

Temperature-dependent surface potentials of fluorinated alkanethiolate self-assembled monolayers

Self-assembled monolayers (SAMs) consisting of twenty-two carbon, methyl-terminated alkanethiolates adsorbed on vapor-deposited gold have been fluorinated in vacuum using an effusive F atom source. The reactive uptake of fluorine as a function of F atom exposure was calibrated using X-ray photoelectron spectroscopy. The surface potentials (V_s) of SAMs that were fluorinated to different degrees were measured as a function of temperature using a high-sensitivity vibrating probe electrostatic voltmeter. The surface potential grew increasingly negative with increasing fluorine uptake, reflecting the charge asymmetry that is induced in the alkanethiolate chains as a result of the substitution of fluorine for hydrogen. The V_s of the most highly fluorinated SAMs displayed a negative temperature dependence. This observation may be indicative of a pyroelectric effect in these monolayers although a definitive conclusion awaits further measurements.     

Dual desorption electrospray ionization-laser desorption ionization mass spectrometry on a common nanoporous alumina platform for enhanced shotgun proteomic analysis

A gold coated nanoporous alumina surface was used for dual ionization mode mass spectrometric analysis using desorption electrospray ionization (DESI) and laser desorption ionization (LDI). DESI and LDI mass spectrometry (MS) from the nanoporous alumina surface were compared with conventional electrospray ionization (ESI) mass spectrometry and matrix assisted laser desorption ionization (MALDI) for analysis of tryptic digests of proteins. Combined use of DESI and LDI offer greater peptide coverage than either method alone and comparable peptide coverage as with dual MALDI and ESI. This dual ionization technique using a common platform with same sample spot demonstrates a potential time and cost-effective tool for improved shotgun proteomic analysis.     

Protein arrays on patterned porous gold substrates interrogated with mass spectrometry: detection of peptides in plasma

Methyl- and carboxy-terminated self-assembled monolayers (SAMs) were custom-patterned on porous gold substrates with equipment commonly used to print protein arrays, without complex surface chemistry protocols. Proteins were covalently immobilized on hydrophilic carboxy-terminated SAM spots, while the remainder of the surface was superhydrophobic due to the roughened gold surface and the methyl-terminated SAM. The resistance of these patterns to biofouling and the effective containment of MALDI matrix solution within the hydrophilic spot made these surfaces amenable to analyzing protein-peptide binding with mass spectrometry. A model system of the affinity peptides HA, cmyc, and V5 and their corresponding antibodies was used to demonstrate the utility of the patterned porous gold. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectra and images obtained reflected the effective capture of the affinity peptides directly from spiked bovine plasma.     

Analysis of lipids: metal oxide laser ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for lipid analysis; however, one of the drawbacks of this technique is matrix interference peaks at low masses. Metal oxide surfaces are described here for direct, matrix-free analysis of small (MW < 1000 Da) lipid compounds, without interferences in the resulting spectra from traditional matrix background peaks. Spectra from lipid standards produced protonated and sodiated molecular ions. More complex mixtures including vegetable oil shortening and lipid extracts from bacterial and algal sources provided similar results. Mechanistic insight into the mode of ionization from surface spectroscopy, negative ion mass spectrometry, and stable isotope studies is also presented. The metal oxide system is compared to other reported matrix-free systems.     

Mass spectrometry of acoustically levitated droplets

Containerless sample handling techniques such as acoustic levitation offer potential advantages for mass spectrometry, by eliminating surfaces where undesired adsorption/desorption processes can occur. In addition, they provide a unique opportunity to study fundamental aspects of the ionization process as well as phenomena occurring at the air-droplet interface. Realizing these advantages is contingent, however, upon being able to effectively interface levitated droplets with a mass spectrometer, a challenging task that is addressed in this report. We have employed a newly developed charge and matrix-assisted laser desorption/ionization (CALDI) technique to obtain mass spectra from a 5-microL acoustically levitated droplet containing peptides and an ionic matrix. A four-ring electrostatic lens is used in conjunction with a corona needle to produce bursts of corona ions and to direct those ions toward the droplet, resulting in droplet charging. Analyte ions are produced from the droplet by a 337-nm laser pulse and detected by an atmospheric sampling mass spectrometer. The ion generation and extraction cycle is repeated at 20 Hz, the maximum operating frequency of the laser employed. It is shown in delayed ion extraction experiments that both positive and negative ions are produced, behavior similar to that observed for atmospheric pressure matrix-assisted laser absorption/ionization. No ion signal is observed in the absence of droplet charging. It is likely, although not yet proven, that the role of the droplet charging is to increase the strength of the electric field at the surface of the droplet, reducing charge recombination after ion desorption.     

Adjustable fragmentation in laser desorption/ionization from laser-induced silicon microcolumn arrays

Laser-induced silicon microcolumn arrays (LISMA) were developed as matrix-free substrates for soft laser desorption/ionization mass spectrometry (SLDI-MS). When low-resistivity silicon wafers were irradiated in air, sulfur hexafluoride, or water environment with multiple pulses from a 3 x omega mode-locked Nd:YAG laser, columnar structures were formed on the surface. The radii of curvature of the column tips varied with the processing environment, ranging from approximately 120 nm in water, to <1 mum in SF6, and to approximately 2 mum in air. In turn, these microcolumn arrays were used as matrix-free soft laser desorption substrates. In SLDI-MS experiments with a nitrogen laser, the microcolumn arrays obtained in water environment readily produced molecular ions for peptides and synthetic polymers at low laser fluence. These surfaces demonstrated the best ion yield among the three arrays. The threshold laser fluence and ion yield were comparable to those observed in matrix-assisted laser desorption/ionization. Low-femtomole sensitivity and approximately 6000 Da mass range were achieved. At elevated laser fluence, efficient in-source decay was observed and extensive peptide sequence information was extracted from the resulting mass spectra. The versatility of LISMA was attributed to confinement effects due to the submicrometer morphology and to the surface, thermal, and optical properties of processed silicon.     

Laser desorption and matrix-assisted laser desorption/ionization mass spectrometry of 29-kDa Au:SR cluster compounds

Positive and negative ions generated by laser-based ionization methods from three gold:thiolate cluster compounds are mass analyzed by time-of-flight mass spectrometry. The three compounds have similar inorganic core masses ( approximately 29 kDa, approximately 145 Au atoms) but different n-alkanethiolate ligands associated with each cluster compound (Au:SR, R = butane, hexane, dodecane). Irradiation of neat films (laser desorption/ionization) and films generated by dilution of the cluster compounds in an organic acid matrix (matrix-assisted laser desorption/ionization) with a nitrogen laser (337 nm) produced distinct ion abundances that are relevant to different structural aspects of the cluster compound. Laser desorption/ionization of neat Au:SR compound films produces ions consistent with the inorganic core mass (i.e., devoid of original hydrocarbon content). Matrix-assisted laser desorption/ionization produces either ions with m/z values consistent with the core mass of the cluster compounds or ions with m/z values consistent with the approximate molecular weight of the cluster compounds, depending on ionization conditions. The ion abundances, and ionization conditions under which they are detected, provide insight into desorption/ionization processes for these unique cluster compounds as well as other analytes typically studied by matrix-assisted laser desorption/ionization.     

Lectin-based affinity capture for MALDI-MS analysis of bacteria

Immobilized lectins have now been incorporated into affinity surfaces that can be used to isolate broad classes of samples for mass spectrometric analysis. A carbohydrate and a bacterial species that displays the carbohydrate binding motif were isolated and concentrated out of solutions containing salt, urea, buffers, and other contaminants that are deleterious to MALDI mass spectrometry. Concanavalin A was immobilized to a gold foil via a self-assembled monolayer. Samples in phosphate buffer or urine were applied to the capture surface and allowed to interact. The capture surface was then washed to remove salts and other unbound components and subjected to matrix-assisted laser desorption/ionization on a time-of-flight mass spectrometer. The lectin-derivatized surface allowed samples to be concentrated and readily characterized at relatively low levels.     

Ordered porous alumina geometries and surface metals for surface-assisted laser desorption/ionization of biomolecules: possible mechanistic implications of metal surface melting

Platinum- or gold-coated porous alumina with submicrometer structures is a potential substrate for surface-assisted laser desorption/ionization (SALDI) mass spectrometry of biomolecules, not requiring sample matrixes. In this study, a highly ordered porous alumina substrate was fabricated to study the geometric factors allowing good SALDI performance. Evaluation was based on the signal-to-noise ratio of protonated angiotensin I ions in the mass spectrum obtained by 337-nm ultraviolet laser irradiation. Varying the geometries, including pore densities and diameters, revealed the laser intensity required to generate ions to be related to surface porosity. Surface platinum was melted upon laser irradiation at the fluence sufficient to generate peptide ions as confirmed by scanning electron microscopy. Moreover, a thin (5-20 nm) platinum coat requires a low intensity of laser light for desorption/ionization. Considering the size effect on the melting of metals, our findings suggest the surface platinum melting to be involved in ion generation from this SALDI substrate type. Indeed, tantalum, which has a higher melting point, required more laser fluence to generate ions. The porous alumina layer beneath surface metals probably worked as a thermal insulator. This double-layer-type substrate allowed ionization of angiotensin I and verapamil at low femtomole levels. Moreover, small proteins and glycoproteins such as 24-kDa trypsinogen and 15-kDa ribonuclease B could be ionized with sufficient sensitivity on this target. Taking advantage of matrix-free methods, concentrating the sample solution in the target concavity or widening the laser beam focus enhanced the signal-to-noise ratio for analyte ions in the mass spectrum. Activity is maintained for months in air.     

Analysis of self-assembled monolayers on gold surfaces using direct analysis in real time mass spectrometry

Mass spectrometry was performed on self-assembled monolayers (SAMs) of dodecanethiol on gold using the new direct analysis in real time (DART) ionization technique. Observed peaks for the SAMs included monomers, dimers, and trimers of the SAM molecules, with the dimer and trimer relative peak heights enhanced as compared to the spectra for neat dodecanethiol. The possibility that the observed peaks were due to residual (noncovalently bonded) material on the surface was tested by attempting to observe residual dodecanol. No peaks corresponding to dodecanol were observed. These results indicate that DART is an excellent ionization method for the direct and unambiguous mass analysis of chemical species in self-assembled monolayers.     

FAB mass spectrometry of Au25(SR)18 nanoparticles

The molecular ion of the nanoparticle Au 25(SCH 2CH 2Ph) 18 (A 25(SR) 18) is observed at 7394 Da in fast atom bombardment (FAB, Xe atoms) ionization mass spectrometry using a 3-nitrobenzyl alcohol matrix. A distinctive pattern of positive fragment ions is evident in the mass interval 5225-7394 Da, where peaks are seen for successive mass losses equivalent to R 2S entities. Because the Au 25(SCH 2CH 2Ph) 18 nanoparticle structure is crystallographically known to consist of a centered Au 13 icosahedral core surrounded by six Au 2(SR) 3 semirings, the R 2S loses are proposed to represent serial rearrangements and decompositions of the semiring structures. Mass losses equivalent to R 2S 2 and R 2 entities also appear at the lower end of this mass interval. The most intense spectral peak, at m/ z = 5246 Da, is assigned to the fragment Au 25S 10, from which all of the CH 2CH 2Ph organic units have been cleaved but from which no gold atoms have been lost. A different pattern of fragmentation is observed at lower masses, producing ions corresponding to serial losses of one gold atom and varied numbers of sulfur atoms, which continues down to a Au 9S 2 fragment. FAB mass spectra of the Au nanoparticle are much easier to interpret than laser desorption/ionization spectra, but they show more extensive fragmentation than do electrospray and low laser pulse intensity MALDI spectra. The loss of R 2S fragmentation in FAB is distinctive and unlike that seen in the other ionization modes. The FAB spectrum for the nanoparticle Au 25(S(CH 2) 9CH 3) 18 is also reported; its fragmentation parallels that for Au 25(SCH 2CH 2Ph) 18, implying that this nanoparticle has the same surprising stellated (staples) structure.     

Charge reduction electrospray mass spectrometry

A new mass spectrometric technique, charge reduction electrospray mass spectrometry (CREMS), allowing the analysis of complex mixtures of biological molecules is described. The charge state of ions produced by electrospray ionization may be reduced in a controlled manner to yield predominantly singly charged ions through reactions with bipolar (i.e., both positively and negatively charged) ions generated using a 210Po alpha particle source. The electrospray-generated multiply charged ions undergo charge reduction in a "neutralization chamber" positioned before the entrance nozzle to the mass spectrometer. The ions are detected using a commercial orthogonal electrospray time-of-flight mass spectrometer, although the neutralization chamber can be adapted to virtually any mass analyzer. The CREMS results obtained exhibit a signal intensity drop-off with increasing oligonucleotide size similar to that observed with matrix-assisted laser desorption/ionization mass spectrometry. Proton-transfer reactions were found to be responsible for reducing charge on proteins and oligonucleotides in both positive and negative ion mode.     

Mass spectrometric analysis of benzoylated sialooligosaccharides and differentiation of terminal alpha 2-->3 and alpha 2-->6 sialogalactosylated linkages at subpicomole levels

Perbenzoylated sialooligosaccharides were found to be stable derivatives, giving intense signals during the matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis in the positive-ion mode. Terminal Neu5NAc alpha 2-->3 and alpha 2-->6Gal units of oligosaccharides undergo characteristic structural changes during benzoylation, yielding easily recognizable mass spectral patterns. Subpicomole carbohydrate samples were successfully benzoylated and analyzed through MALDI mass spectrometry.     

Matrix-free laser desorption/ionization-mass spectrometry using self-assembled germanium nanodots

A novel ionization platform for matrix-free laser desorption/ionization-mass spectrometry (LDI-MS) was developed using self-assembled germanium nanodots (GeNDs) of uniform size (approximately 150 - 200-nm width and approximately 50-nm height) grown on a silicon wafer produced by molecular beam epitaxy. The performance of LDI-MS using GeNDs (GeND-MS) was investigated through measurements of a broad range of analytes, including peptides, proteins, synthetic oligomers, and polymer additives. Mass spectra of tryptic digests were clearly observed even for the mass range lower than m/z 800 without obstructive peaks. A detection limit of subfemtomole level was achieved for angiotensin-I. The upper limit of detectable mass range was approximately 17 kDa (myoglobin). GeND-MS also has potential for application to the characterization of industrial compounds. Almost accurate molecular weight distribution was obtained for a nonionic surfactant (Triton X-100) and for poly(ethylene glycol) oligomer. Furthermore, a brominated flame retardant, tetrabromobisphenol-A bis(2,3-dibromopropyl ether), was successfully ionized with less fragmentation, a result not obtainable by matrix-assisted laser desorption/ionization-mass spectrometry or desorption/ionization on porous silicon-mass spectrometry.     

Fast screening of low molecular weight compounds by thin-layer chromatography and "on-spot" MALDI-TOF mass spectrometry

Fast screening of low-MW compounds is performed by thin-layer chromatography (TLC) followed by direct on-spot matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification with nearly "matrix-free" mass spectra using an UV-absorbing ionic liquid matrix. Owing to minimal background ions from the proton donor triethylamine/alpha-cyano-4-hydroxycinnamic acid ionic liquid matrix, three arborescidine alkaloids, the anesthesics levobupivacaine and mepivacaine, and the antibiotic tetracycline were readily characterized most frequently by the MS detection of their protonated molecules. The technique is fast and sensitive, requires little sample preparation and manipulation, and is therefore suitable for fast screening with TLC separation and MS identification of low-MW compounds, with potential applications in areas such as phytochemistry, synthetic chemistry, and product manufacturing quality monitoring.     

Functionalized magnetic nanoparticles for small-molecule isolation, identification, and quantification

Functionalized magnetic nanoparticles (MNPs) were synthesized to serve as laser desorption/ionization elements as well as solid-phase extraction probes for simultaneous enrichment and detection of small molecules in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Two laser-absorbing matrices were each conjugated onto MNP to give MNP@matrix which provided high ionization efficiency and background-free detection in MS leading to unambiguous identification of target small molecules in a complex mixture. MNP@matrix was demonstrated to serve as a general matrix-free additive in MALDI-TOF MS analysis of structurally distinct small molecules. Also, MNP@matrix provides a simple, rapid, and reliable quantitative assay for small molecules by mass without either the use of an internal standard or an isotopic labeling tag. Furthermore, the affinity extraction of small molecules from complex biofluid was achieved by probe protein-conjugated MNP@matrix without laborious purification. We demonstrated that a nanoprobe-based assay is a cost-effective, rapid, and accurate platform for robotic screening of small molecules.     

Nanostructured diamond-like carbon on digital versatile disc as a matrix-free target for laser desorption/ionization mass spectrometry

A nanostructured diamond-like carbon (DLC) coated digital versatile disk (DVD) target is presented as a matrix-free sample support for application in laser desorption/ionization mass spectrometry (LDI-MS). A large number of vacancies, defects, relative sp(2) carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of 305-330 nm (nitrogen laser, 337 nm). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites. Carbohydrates and amino acids are analyzed as sodium and potassium adducts. Peptides are detectable in their protonated forms, which avoid the extra need of additives for ionization. A bovine serum albumin (BSA) digest is analyzed to demonstrate the performance for peptide mixtures, coupled with the material-enhanced laser desorption/ionization (MELDI) approach. The detection limit of the described matrix-free target is investigated to be 10 fmol/microL for [Glu(1)]-fibrinopeptide B (m/z 1570.6) and 1 fmol/microL for L-sorbose (Na(+) adduct). The device does not require any chemical functionalization in contrast to other matrix-free systems. The inertness of DLC provides longer lifetimes without any deterioration in the detection sensitivity. Broad applicability allows high performance analysis in metabolomics and peptidomics. Furthermore the DLC coated DVD (1.4 GB) sample support is used as a storage device for measured and processed data together with sampling on a single device.     

Atmospheric pressure laser-induced acoustic desorption chemical ionization fourier transform ion cyclotron resonance mass spectrometry for the analysis of complex mixtures

We present a novel nonresonant laser-based matrix-free atmospheric pressure ionization technique, atmospheric pressure laser-induced acoustic desorption chemical ionization (AP/LIAD-CI). The technique decouples analyte desorption from subsequent ionization by reagent ions generated from a corona discharge initiated in ambient air or in the presence of vaporized toluene as a CI dopant at room temperature. Analyte desorption is initiated by a shock wave induced in a titanium foil coated with electrosprayed sample, irradiated from the rear side by high-energy laser pulses. The technique enables facile and independent optimization of the analyte desorption, ionization, and sampling events, for coupling to any mass analyzer with an AP interface. Moreover, the generated analyte ions are efficiently thermalized by collisions with atmospheric gases, thereby reducing fragmentation. We have coupled AP/LIAD-CI to ultrahigh-resolution FT-ICR MS to generate predominantly [M + H](+) or M(+?) ions to resolve and identify thousands of elemental compositions from organic mixtures as complex as petroleum crude oil distillates. Finally, we have optimized the AP/LIAD CI process and investigated ionization mechanisms by systematic variation of placement of the sample, placement of the corona discharge needle, discharge current, gas flow rate, and inclusion of toluene as a dopant.     

Laser desorption/ionization time-of-flight mass spectrometry on sol-gel-derived 2,5-dihydroxybenzoic acid film

This work presents a novel method for direct desorption/ ionization of analytes from sol-gel-derived film. 2,5-Dihydroxy benzoic acid (DHB), a common MALDI matrix, was incorporated into a sol-gel polymeric structure. The sol-gel-derived DHB thin film can assist the mass analysis of analytes by laser desorption/ionization, with a matrix interference-free background in the mass spectra. The sol-gel-derived film can function as an energy absorber during laser irradiation because it contains DHB molecules. Furthermore, laser irradiation with normal laser power (70-110 microJ) is not likely to generate any background ions from this sol-gel-DHB derived film. The samples were prepared straightforwardly. After a thin film was formed on a Parafilm membrane from the sol-gel-derived DHB solution coating, the sample solution was directly added to the top of the film, for laser desorption/ ionization mass analysis. The analyte signals were homogeneously obtained on the sol-gel-derived DHB film. Experimental results show that the optimum concentrations of DHB incorporated in the sol-gel solution were between 7,500 ppm and 10,000 ppm, providing a matrix interference-free background. Analytes, including small proteins, peptides, amino acids, and small organics, were used to demonstrate the effectiveness of the proposed method. However, a higher laser power (> 110 microJ) than normal was required to desorb small proteins from the sol-gel-derived DHB film. Therefore, a few matrix ions desorbed from the thin film were generated during laser irradiation. The detection limit for both small molecules and proteins, using this sol-gel-assisted laser desorption/ ionization (SGALDI) mass spectrometry (MS), was as low as 81 fmol. However, a mass spectrometer with cutoff-mass selection could detect 8.1 fmol of cytochrome c. The largest analyte observed by the SGALDI-MS in this study was myoglobin.