High sensitivity and analyte capture with desorption/ionization mass spectrometry on silylated porous silicon

Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.     

Surfactant-enhanced desorption/ionization on silicon mass spectrometry

Perfluorinated surfactants are demonstrated to dramatically enhance desorption/ionization on fluorinated silicon (DIOS) mass spectrometry. Perfluorooctanesulfonic acid improved the signal-to-noise ratio of tryptic digests and gave a 3-fold increase in the number of peptides identified. Similar results were also obtained using perfluoroundecanoic acid; yet among the seven different surfactants tested, controls such as nonfluorinated sodium dodecyl sulfate or fluorinated molecules with minimal surfactant activity did not enhance the signal. The same surfactants also enhanced the DIOS-MS signal of amino acids, carbohydrates, and other small organic compounds. The signal enhancement may be facilitated by the high surface activity of the perfluorinated surfactants on the fluorinated silicon surfaces allowing for a higher concentration of analyte to be absorbed.     

A quantitative model of ultraviolet matrix-assisted laser desorption/ionization including analyte ion generation

A quantitative model of ionization in ultraviolet matrix-assisted laser desorption/ionization (Knochenmuss, R. J. Mass Spectrom. 2002, 37, 867) is extended to include secondary ion-molecule reactions. Matrix-to-analyte charge-transfer reaction kinetics are described by a hard-sphere Arrhenius expression. The activation energy is derived from the reaction exoergicity using a nonlinear free energy relationship. The approach is applied to the specific case of proton-transfer reactions. With no adjustable parameters, the model correctly predicts the existence and characteristics of the matrix and analyte suppression effects, the shapes of the two-pulse time-delayed yield curves, and the dependence of analyte yields on laser fluence, molecular weight, relative concentrations, and reaction exoergicity.     

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.     

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.     

Porous silicon as a versatile platform for laser desorption/ionization mass spectrometry

Desorption/ionization on porous silicon mass spectrometry (DIOS-MS) is a novel method for generating and analyzing gas-phase ions that employs direct laser vaporization. The structure and physicochemical properties of the porous silicon surfaces are crucial to DIOS-MS performance and are controlled by the selection of silicon and the electrochemical etching conditions. Porous silicon generation and DIOS signals were examined as a function of silicon crystal orientation, resistivity, etching solution, etching current density, etching time, and irradiation. Pre-and postetching conditions were also examined for their effect on DIOS signal as were chemical modifications to examine stability with respect to surface oxidation. Pore size and other physical characteristics were examined by scanning electron microscopy and Fourier transform infrared spectroscopy, and correlated with DIOS-MS signal. Porous silicon surfaces optimized for DIOS response were examined for their applicability to quantitative analysis, organic reaction monitoring, post-source decay mass spectrometry, and chromatography.     

Targeted analyte detection by standard addition improves detection limits in matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) has proven an effective tool for fast and accurate determination of many molecules. However, the detector sensitivity and chemical noise compromise the detection of many invaluable low-abundance molecules from biological and clinical samples. To challenge this limitation, we developed a targeted analyte detection (TAD) technique. In TAD, the target analyte is selectively elevated by spiking a known amount of that analyte into the sample, thereby raising its concentration above the noise level, where we take advantage of the improved sensitivity to detect the presence of the endogenous analyte in the sample. We assessed TAD on three peptides in simple and complex background solutions with various exogenous analyte concentrations in two MALDI matrices. TAD successfully improved the limit of detection (LOD) of target analytes when the target peptides were added to the sample in a concentration close to optimum concentration. The optimum exogenous concentration was estimated through a quantitative method to be approximately equal to the original LOD for each target. Also, we showed that TAD could achieve LOD improvements on an average of 3-fold in a simple and 2-fold in a complex sample. TAD provides a straightforward assay to improve the LOD of generic target analytes without the need for costly hardware modifications.     

Enzyme kinetics by directly imaging a porous silicon microfluidic reactor using desorption/ionization on silicon mass spectrometry

Enzyme kinetics were obtained in a porous silicon microfluidic channel by combining an enzyme and substrate droplet, allowing them to react and deposit a small amount of residue on the channel walls, and then analyzing this residue by directly ionizing the channel walls using a matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) laser source. The porous silicon of the channel walls functions in a manner analogous to the matrix in MALDI-MS, and is referred to as a desorption/ionization on silicon mass spectrometry (DIOS-MS) target when used in this configuration. Mass spectrometry signal intensity of substrate residue correlates with relative concentration, and position in the microchannel correlates with time, thus allowing determination of kinetic parameters. The system is especially suitable for initial reaction velocity determination. This microreactor is broadly applicable to time-resolved kinetic assays as long as at least one substrate or product of the reaction is ionizable by DIOS-MS.     

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.     

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.     

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.     

Ordered silicon nanocavity arrays in surface-assisted desorption/ionization mass spectrometry

We report here a simple method to generate ordered nanocavity arrays on a Si wafer and use it in surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). A close-packed SiO2 nanosphere array was first deposited on a low-resistivity Si wafer using a convective self-assembly method. The nanoparticle array was then used as a mask in a reactive ion etching (RIE) process to selectively remove portions of the Si surface. Subsequent sonication removed those physically adsorbed SiO2 nanoparticles and exposed an ordered nanocavity array underneath. The importance of this approach is its capability of systematically varying surface geometries to achieve desired features, which makes detailed studies of the impacts of surface features on the desorption/ionization mechanism feasible. We demonstrated that the in-plane width and out-of-plane depth of the cavities were adjustable by varying etching times, and the intercavity spacing was controllable by varying the number of particle layers deposited. MS detection of small peptides on these substrates showed comparable sensitivity to conventional porous Si substrates (DIOS, desorption/ ionization on porous silicon). The desorption and ionization efficiency of these roughened surfaces exhibited a nonmonotonic relationship to the increased total surface area. Several possible factors contributing to the observed phenomenon are speculated upon. The application of this arrayed surface in metabolite detection of Arabidopsis thaliana root extracts is also demonstrated.     

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.     

Infrared laser desorption/ionization on silicon

Laser desorption/ionization from a single-crystal silicon surface was performed using a laser operating in the 3-microm region of the mid-infrared. Analyte molecules up to 6 kDa were ionized with no added matrix. As with ultraviolet desorption/ionization from porous silicon (DIOS), IR laser desorption from silicon does not produce matrix ions that can interfere with analysis of low-mass analytes. However, in contrast to UV DIOS, silicon porosity or roughness is not required for ionization using an IR laser. Mass spectra were obtained in the wavelength range between 2.8 and 3.5 microm, which is consistent with energy absorption by a hydrogen-bonded OH group. A mechanism based on desorption of adsorbed solvent molecules is postulated.     

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.     

Desorption/ionization on porous silicon mass spectrometry studies on pentose-borate complexes

Desorption/ionization on porous silicon mass spectrometry (DIOS-MS) was used to investigate the binding affinities between aldopentose isomers and boron. Boron has been recognized for its importance in pentose synthesis and stabilization in prebiotic conditions. Boron may also account for the fact that ribose, among other aldopentoses, is the favored building block in RNA synthesis. This research started with the detection of aldopentoses in the positive mode through cationization and the aldopentose-borate complexes in the negative mode. Then two competition schemes, one using a pentose structure analogue and the other using 13C-labeled ribose, were designed to compare the relative binding affinities of four aldopentoses (xylose, lyxose, arabinose, and ribose) to boron. Both approaches determined the binding preference to be ribose > lyxose > arabinose > xylose. This work illustrates the potential of DIOS-MS in the analyses of nonvolatile, small molecules in delicate chemical equilibria. Without externally introduced matrices, background signals are not a limiting factor. Furthermore, the possible dramatic change of pH associated with the matrix introduction, which may disturb the equilibria of interest, is avoided.     

Quantitative analysis with desorption/ionization on silicon mass spectrometry using electrospray deposition

Desorption/ionization on silicon mass spectrometry (DIOS-MS) is demonstrated as a quantitative analytical tool when coupled to electrospray deposition (ESD). In this study, we illustrate the utility of DIOS-MS in the quantitative analysis of a peptide and two amino acids with deuterated and structural analogues used as internal standards. An important feature of this approach is the incorporation of ESD to improve sample homogeneity across the porous silicon surface. ESD allowed for a marked improvement in quantitative analysis due to its applicability to LC-DIOS, and because of the absence of matrix, sample can be deposited at very low flow rates (150 nL/min). Experiments comparing the traditional dried droplet and ESD methods show that ESD samples exhibit significantly improved quantitation and much higher sample-to-sample reproducibility.     

Iron oxide nanomatrix facilitating metal ionization in matrix-assisted laser desorption/ionization mass spectrometry

The significance and epidemiological effects of metals to life necessitate the development of direct, efficient, and rapid method of analysis. Taking advantage of its simple, fast, and high-throughput features, we present a novel approach to metal ion detection by matrix-functionalized magnetic nanoparticle (matrix@MNP)-assisted MALDI-MS. Utilizing 21 biologically and environmentally relevant metal ion solutions, the performance of core and matrix@MNP against conventional matrixes in MALDI-MS and laser desorption ionization (LDI) MS were systemically tested to evaluate the versatility of matrix@MNP as ionization element. The matrix@MNPs provided 20- to >100-fold enhancement on detection sensitivity of metal ions and unambiguous identification through characteristic isotope patterns and accurate mass (<5 ppm), which may be attributed to its multifunctional role as metal chelator, preconcentrator, absorber, and reservoir of energy. Together with the comparison on the ionization behaviors of various metals having different ionization potentials (IP), we formulated a metal ionization mechanism model, alluding to the role of exciton pooling in matrix@MNP-assisted MALDI-MS. Moreover, the detection of Cu in spiked tap water demonstrated the practicability of this new approach as an efficient and direct alternative tool for fast, sensitive, and accurate determination of trace metal ions in real samples.