Layer-by-layer self-assembled mutilayer films of gold nanoparticles for surface-assisted laser desorption/ionization mass spectrometry

Layer-by-layer (LBL) self-assembled multilayer films of gold nanoparticles (AuNPs) on a silicon wafer were demonstrated to be promising substrates for surface-assisted laser desorption/ionization (SALDI) mass spectrometry (MS) of peptides and environmental pollutants for the first time. LBL multilayer films, (AuNPs/PAHC)n, consisting of alternating layers of ammonium citrate capped AuNPs and poly(allylamine hydrochloride) (PAHC) were prepared on a silicon surface. Silicon plates with aggregated AuNPs were more suitable than those with dispersed AuNPs for the SALDI-MS of peptides. The number of particle layers had a significant effect on the laser desorption/ionization of angiotensin I; the peak intensity of the peptide (molecular ion amount) increased with an increase in the number of layers of AuNPs. As a result, the (AuNPs/PAHC)5 multilayer films increased the sensitivity of the angiotensin I to subfemtomoles and raised the useful analyte mass range, thus making it possible to detect small proteins (a 12 kDa cytochrome c). The signal enhancement when using (AuNPs/PAHC)5 may be due to (i) the high absorption of the UV laser light at 337 nm by the AuNP layers, (ii) the low thermal conductivity due to the AuNPs being covered with a thin monolayer of PAHC, and (iii) the increase in the surface roughness (approximately 100 nm) with the number of AuNP layers. Thus, laser-induced rapid high heating of AuNPs for effective desorption/ionization of peptides is possible. In addition, it was found that (AuNPs/PAHC)5 could be used to extract environmental pollutants (pyrene and dimethyldistearylammonium chloride) from very dilute aqueous solutions with concentrations less than 10(-10) mg/mL, and the analytes trapped in the LBL film could be identified by introducing the film directly into the SALDI mass spectrometer without needing to elute the analytes out of the film.     

Dynamic electrowetting on nanofilament silicon for matrix-free laser desorption/ionization mass spectrometry

Dynamic electrowetting on nanostructured silicon surfaces is demonstrated as an effective method for improving detection sensitivity in matrix-free laser desorption/ionization mass spectrometry. Without electrowetting, silicon surfaces comprising dense fields of oriented nanofilaments are shown to provide efficient ion generation and high spectral peak intensities for deposited peptides bound to the nanofilaments through hydrophobic interactions. By applying an electrical bias to the silicon substrate, the surface energy of the oxidized nanofilaments can be dynamically controlled by electrowetting, thereby allowing aqueous buffer to penetrate deep into the nanofilament matrix. The use of electrowetting is shown to result in enhanced interactions between deposited peptides and the nanofilament silicon surface, with improved signal-to-noise ratio for detected spectral peaks. An essential feature contributing to the observed performance enhancement is the open-cell nature of the nanofilament surfaces, which prevents air from becoming trapped within the pores and limiting solvent penetration during electrowetting. The combination of nanofilament silicon and dynamic electrowetting is shown to provide routine detection limits on the order of several attomoles for a panel of model peptides.     

Analysis of adenosine triphosphate and glutathione through gold nanoparticles assisted laser desorption/ionization mass spectrometry

This paper describes the use of aptamer-modified gold nanoparticles (Apt-AuNPs) as selective probes and AuNPs as the surface-assisted laser desorption/ionization (SALDI) matrixes for the determination of adenosine triphosphate (ATP) by mass spectrometry (MS). The aptamers were covalently attached to the surface of AuNPs to form Apt-AuNPs that provided selectivity toward ATP. However, Apt-AuNPs are less efficient laser desorption/ionization (LDI) matrixes when compared to AuNPs. By using Apt-AuNPs as selective probes and AuNPs as LDI matrixes, the MS approach provided the limit of detection (LOD) for ATP at a signal-to-noise ratio of 3 of 0.48 microM. When compared to conventional organic matrixes (e.g., 2,5-dihydroxybenzoic acid), AuNPs as LDI matrixes provide a number of advantages, including ease of preparation, selectivity, sensitivity, and repeatability. Sequential analysis of ATP and GSH in human cell lysates by SALDI with negative and positive MS modes, respectively, using Apt-AuNPs and AuNPs has been demonstrated. The present results demonstrate the practicality of the approach for monitoring the bioactivity of cells through determinations of the concentrations of ATP and GSH.     

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.     

Matrix-implanted laser desorption/ionization mass spectrometry

The implantation of low-velocity massive gold clusters is shown to be a method of choice for homogeneous incorporation of a metallic matrix into the near-surface region of a solid biopolymer for subsequent laser desorption/ionization (LDI) MS analysis. Matrix implanted (MI)LDI spectra from cluster-implanted pure test peptide or tissue exhibit molecular ion peaks similar to those observed by matrix-assisted LDI. Moreover, the ion emission is very reproducible from any spot on the surface of these test samples. MILDI promises to be a powerful technique for mass spectrometric analysis of native biological samples as demonstrated by the first results on rat brain tissues.     

Aptamer-enhanced laser desorption/ionization for affinity mass spectrometry

The thrombin-binding DNA aptamer was used for affinity capture of thrombin in MALDI-TOF-MS. The aptamer was covalently attached to the surface of a glass slide that served as the MALDI surface. Results show that thrombin is retained at the aptamer-modified surface while nonspecific proteins, such as albumin, are removed by rinsing with buffer. Upon application of the low-pH MALDI matrix, the G-quartet structure of the aptamer unfolds, releasing the captured thrombin. Following TOF-MS analysis, residual matrix and protein can be washed from the surface, and buffer can be applied to refold the aptamers, allowing the surface to be reused. Selective capture of thrombin from mixtures of thrombin and albumin and of thrombin and prothrombin from human plasma was demonstrated. This simple approach to affinity capture, isolation, and detection holds potential for analysis, sensing, purification, and preconcentration of proteins in biological fluids.     

Discrimination between bacterial spore types using time-of-flight mass spectrometry and matrix-free infrared laser desorption and ionization

We demonstrate that molecular ions with mass-to-charge ratios (m/z) ranging from a few hundred to 19 050 can be desorbed from whole bacterial spores using infrared laser desorption and no chemical matrix. We have measured the mass of these ions using time-of-flight mass spectrometry and we observe that different ions are desorbed from spores of Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Bacillus niger. Our results raise the possibility of identifying microorganisms using mass spectrometry without conventional sample preparation techniques such as the addition of a matrix. We have measured the dependence of the ion yield from B. subtilis on desorption wavelength over the range 3.05-3.8 microm, and we observe the best results at 3.05 microm. We have also generated mass spectra from whole spores using 337-nm ultraviolet laser desorption, and we find that these spectra are inferior to spectra generated with infrared desorption. Since aerosol analysis is a natural application for matrix-free desorption, we have measured mass spectra from materials such as ragweed pollen and road dust that are likely to form a background to microbial aerosols. We find that these materials are readily differentiated from bacterial spores.     

Comprehensive assignment of mass spectral signatures from individual Bacillus atrophaeus spores in matrix-free laser desorption/ionization bioaerosol mass spectrometry

We have fully characterized the mass spectral signatures of individual Bacillus atrophaeus spores obtained using matrix-free laser desorption/ionization bioaerosol mass spectrometry (BAMS). Mass spectra of spores grown in unlabeled, 13C-labeled, and 15N-labeled growth media were used to determine the number of carbon and nitrogen atoms associated with each mass peak observed in mass spectra from positive and negative ions. To determine the parent ion structure associated with fragment ion peaks, the fragmentation patterns of several chemical standards were independently determined. Our results confirm prior assignments of dipicolinic acid, amino acids, and calcium complex ions made in the spore mass spectra. The identities of several previously unidentified mass peaks, key to the recognition of Bacillus spores by BAMS, have also been revealed. Specifically, a set of fragment peaks in the negative polarity is shown to be consistent with the fragmentation pattern of purine nucleobase-containing compounds. The identity of m/z = +74, a marker peak that helps discriminate B. atrophaeus from Bacillus thuringiensis spores grown in rich media is [N1C4H12]+. A probable precursor molecule for the [N1C4H12]+ ion observed in spore spectra is trimethylglycine (+N(CH3)3CH2COOH), which produces a m/z = +74 peak when ionized in the presence of dipicolinic acid. A clear assignment of all the mass peaks in the spectra from bacterial spores, as presented in this work, establishes their relationship to the spore chemical composition and facilitates the evaluation of the robustness of "marker" peaks. This is especially relevant for peaks that have been used to discriminate Bacillus spore species, B. thuringiensis and B. atrophaeus, in our previous studies.     

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.     

Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A novel ionization source for biological mass spectrometry is described that combines atmospheric pressure (AP) ionization and matrix-assisted laser desorption/ionization (MALDI). The transfer of the ions from the atmospheric pressure ionization region to the high vacuum is pneumatically assisted (PA) by a stream of nitrogen, hence the acronym PA-AP MALDI. PA-AP MALDI is readily interchangeable with electrospray ionization on an orthogonal acceleration time-of-flight (oaTOF) mass spectrometer. Sample preparation is identical to that for conventional vacuum MALDI and uses the same matrix compounds, such as alpha-cyano-4-hydroxycinnamic acid. The performance of this ion source on the oaTOF mass spectrometer is compared with that of conventional vacuum MALDI-TOF for the analysis of peptides. PA-AP MALDI can detect low femtomole amounts of peptides in mixtures with good signal-to-noise ratio and with less discrimination for the detection of individual peptides in a protein digest. Peptide ions produced by this method generally exhibit no metastable fragmentation, whereas an oligosaccharide ionized by PA-AP MALDI shows several structurally diagnostic fragment ions. Total sample consumption is higher for PA-AP MALDI than for vacuum MALDI, as the transfer of ions into the vacuum system is relatively inefficient. This ionization method is able to produce protonated molecular ions for small proteins such as insulin, but these tend to form clusters with the matrix material. Limitations of the oaTOF mass spectrometer for singly charged high-mass ions make it difficult to evaluate the ionization of larger proteins.     

Nile Red-adsorbed gold nanoparticle matrixes for determining aminothiols through surface-assisted laser desorption/ionization mass spectrometry

This paper describes the use of Nile Red-adsorbed gold nanoparticles (NRAuNPs) as selective probes and matrixes for the determination of aminothiols through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). The binding of three aminothiols-glutathione (GSH), cysteine (Cys), and homocysteine (HCys)-to the surfaces of these NRAuNPs induces their aggregation, which causes asubsequent changes in their color and fluorescence. Because arginine-a non-thiol amino acid-does not induce such aggregation, it is a straightforward process to use the NRAuNPs to selectively concentrate the aminothiols from a solution containing all four of these analytes; we were able to identify the three aminothiols in the precipitate, and arginine in the supernatant, directly through SALDI-MS measurements. Without using this preconcentration approach, the limits of detection (LODs) at a signal-to-noise ratio of 3 were 1.0, 2.0, and 1.3 microM for GSH, Cys, and HCys, respectively. In comparison, selective concentration using the NRAuNPs provided LODs of 25, 54, and 34 nM, for the determinations of GSH, Cys, and HCys, respectively. NRAuNP matrixes provide a number of advantages over the use of conventional organic matrixes (e.g., 2,5-dihydroxybenzoic acid), such as ease of preparation, selectivity, sensitivity, and repeatability. We validated the applicability of our method through the analyses of GSH in red blood cells and of Cys in plasma; we believe that this approach has great potential for diagnosis.     

Graphite/Liquid mixed matrices for laser desorption/ionization mass spectrometry

A combination of liquid matrix and graphite particulates (2 μm) has been proposed as a method suitable for the laser desorption/ionization mass spectrometry of peptides and proteins (Sunner, J.; et al. Anal. Chem. 1995, 67, 4335). Here we demonstrate the potential of this approach as a straightforward, and very general, method of achieving the ultraviolet laser desorption/ionization of a broad range of intermediate weight analytes. The desorption/ionization mechanism, the influence of preparative procedures, and the breadth of application of this methodology have been investigated. A simple and robust preparative procedure is presented for the analysis of proteins, oligosaccharides, and synthetic polymers. Detection sensitivities are in the femtomole region for lower molecular weight peptides and oligosaccharides. The graphite acts as an energy transfer medium by absorbing the UV radiation, leading to thermal desorption of the liquid matrix and analyte. The liquid matrix was observed to fulfill several important roles. In the case of peptides and proteins, which preferentially form protonated molecular ions, it acts as a protonating agent. It also enhances the signal intensities of cationized species (e.g., polysaccharides and polar polymers) by assisting their desorption. An excess of liquid matrix serves to cool the analyte during the desorption step and minimize decomposition. The presence of liquid matrices increases the sample lifetime at a particular desorption spot, minimizing the time-consuming search for "hot spots". The addition of cationizing salts has been shown to improve the quality of mass spectra obtained for polar polymers and extend the range of materials that can be investigated to include apolar synthetic polymers.     

Role of electrons in laser desorption/ionization mass spectrometry

A nonmetallic sample support for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry enhances the positive ion yield by 2 orders of magnitude and generally affects the charge balance in the desorption plume. We interpret the effects of the target material and of the sample preparation on MALDI mass spectra as a result of photoelectrons emitted upon laser irradiation of a metal target covered by a thin sample layer. These electrons are shown to play an important role in MALDI and laser desorption/ionization because they decrease the yield of positive ions, reduce ions with higher oxidation states, and affect the ion velocity distribution as well as the mass resolution. Understanding the role of these photoelectrons helps to clarify previously obscure aspects of the ion formation mechanism in MALDI.     

Single cell matrix-assisted laser desorption/ionization mass spectrometry imaging

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100?000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.     

Small-molecule analysis with silicon-nanoparticle-assisted laser desorption/ionization mass spectrometry

Silicon nanopowder (5-50 nm) was applied as a matrix for the analysis of small molecules in laser desorption/ionization mass spectrometry. In contrast with conventional matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry, the matrix background interference in the low mass range was significantly reduced. Effects of the particle size and sample preparation procedures on the background mass spectra and the analyte signal intensity have been investigated, and an optimized powder and sample preparation protocol was established. Several surface characterization tools have been applied as well. Both positive mode and negative mode laser desorption/ionization have been applied to different analytes including drugs, peptides, pesticides, acids, and others. Detection limits down to the low femtomole per microliter levels were achieved for propafenone and verapamil drugs. The method developed was found relatively tolerant to salt contamination, which allowed the direct analysis of morphine and propaphenone in untreated urine and triazine herbicides in a soil extract. The new silicon-nanoparticle-assisted laser desorption ionization method was found to be highly selective, which may be due to analyte-dependent precharging in solution, prior to vacuum laser desorption. Some aspects of the charge-transfer mechanism have been studied and discussed. In comparison with standard MALDI matrixes, the silicon nanopowder requires much lower laser fluence (contributing to a reduced background) has much better surface homogeneity, and is more tolerant to salt interference, which makes it an easily applicable practical tool at a potentially low cost.     

Manganese oxide nanoparticle-assisted laser desorption/ionization mass spectrometry for medical applications

Abstract

We prepared and characterized manganese oxide magnetic nanoparticles (d =5.6 nm) and developed nanoparticle-assited laser desorption/ionization (nano-PALDI) mass spectrometry. The nanoparticles had MnO₂ and Mn₂O₃ cores conjugated with hydroxyl and amino groups, and showed paramagnetism at room temperature. The nanoparticles worked as an ionization assisting reagent in mass spectroscopy. The mass spectra showed no background in the low m/z. The nanoparticles could ionize samples of peptide, drug and proteins (approx. 5000 Da) without using matrix, i.e., 2,5-dihydroxybenzoic acid (DHB), 4-hydroxy-α-cinnamic acid (CHCA) and liquid matrix, as conventional ionization assisting reagents. Post source decay spectra by nano-PALDI mass spectrometry will yield information of the chemical structure of analytes.     

Characterization of photooxidized self-assembled monolayers and bilayers by spontaneous desorption mass spectrometry

We show that selected self-assembled monolayers (SAMs) and bilayers are readily characterized by the application of controlled photooxidation and spontaneous desorption mass spectrometry (SDMS) in the negative ion mode. Additionally, SDMS is used to characterize organic and inorganic anionic species adsorbed to the surface of a positively charged SAM surface, 2-aminoethanethiol (AET). Prominent peaks are observed that correspond both to the sulfonate form of each SAM and bilayer and to the anion form of each molecule adsorbed to AET. In addition, fragments of the oxidized thin films were also observed at m/z 80 (SO3-) and 97 (HSO4-). Other prominent fragment peaks more characteristic of the molecule are also seen in the mass spectra.     

Factors affecting quantitative analysis in laser desorption/laser ionization mass spectrometry

Microprobe laser desorption/laser ionization mass spectrometry (microL(2)MS) is a sensitive and selective technique that has proven useful in the qualitative and semiquantitative detection of trace organic compounds, particularly polycyclic aromatic hydrocarbons (PAHs). Recent efforts have focused on developing microL(2)MS as a quantitative method, often by measuring the ratio of signal strength of an analyte to an internal standard. Here, we present evidence of factors that affect these ratios and thus create uncertainty and irreproducibility in quantification. The power and wavelength of the desorption laser, the delay time between the desorption and ionization steps, the power of the ionization laser, and the ionization laser alignment are all shown to change PAH ratios, in some cases by up to a factor of 24. Although changes in the desorption laser parameters and the delay time cause the largest effects, the ionization laser power and alignment are the most difficult parameters to control and thus provide the most practical limitations for quantitative microL(2)MS. Variation in ratios is seen in both synthetic poly(vinyl chloride) membranes and in "real-life" samples of Murchison meteorite powder. Ratios between similar PAHs vary less than those between PAHs that differ greatly in mass and structure. This finding indicates that multiple internal standards may be needed for quantification of samples containing diverse PAHs.     

Ionic liquids as matrixes for matrix-assisted laser desorption/ionization mass spectrometry

Room-temperature ionic liquids are useful as solvents for organic synthesis, electrochemical studies, and separations. We wished to examine whether their high solubalizing power, negligible vapor pressure, and broad liquid temperature range are advantageous if they are used as matrixes for UV-MALDI. Several different ionic matrixes were synthesized and tested, using peptides, proteins, and poly(ethylene glycol) (PEG-2000). All ionic liquids tested have excellent solubilizing properties and vacuum stability compared to other commonly used liquid and solid matrixes. However, they varied widely in their ability to produce analyte gas-phase ions. Certain ionic matrixes, however, produce homogeneous solutions of greater vacuum stability, higher ion peak intensity, and equivalent or lower detection limits than currently used solid matrixes. Clearly, ionic liquids and their more amorphous solid analogues merit further investigation as MALDI matrixes.