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.     

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.     

Atmospheric pressure laser-induced acoustic desorption chemical ionization mass spectrometry for analysis of saturated hydrocarbons

We present atmospheric pressure laser-induced acoustic desorption chemical ionization (AP/LIAD-CI) with O(2) carrier/reagent gas as a powerful new approach for the analysis of saturated hydrocarbon mixtures. Nonthermal sample vaporization with subsequent chemical ionization generates abundant ion signals for straight-chain, branched, and cycloalkanes with minimal or no fragmentation. [M - H](+) is the dominant species for straight-chain and branched alkanes. For cycloalkanes, M(+?) species dominate the mass spectrum at lower capillary temperature (<100 °C) and [M - H](+) at higher temperature (>200 °C). The mass spectrum for a straight-chain alkane mixture (C(21)-C(40)) shows comparable ionization efficiency for all components. AP/LIAD-CI produces molecular weight distributions similar to those for gel permeation chromatography for polyethylene polymers, Polywax 500 and Polywax 655. Coupling of the technique to Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) for the analysis of complex hydrocarbon mixtures provides unparalleled mass resolution and accuracy to facilitate unambiguous elemental composition assignments, e.g., 1754 peaks (rms error = 175 ppb) corresponding to a paraffin series (C(12)-C(49), double-bond equivalents, DBE = 0) and higher DBE series corresponding to cycloparaffins containing one to eight rings. Isoabundance-contoured plots of DBE versus carbon number highlight steranes (DBE = 4) of carbon number C(27)-C(30) and hopanes of C(29)-C(35) (DBE = 5), with sterane-to-hopane ratio in good agreement with field ionization (FI) mass spectrometry analysis, but performed at atmospheric pressure. The overall speciation of nonpolar, aliphatic hydrocarbon base oil species offers a promising diagnostic probe to characterize crude oil and its products.     

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.     

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.     

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.     

Laser desorption/ionization of carbon clusters

Results of mass spectrometric investigations of soot by using the laser desorption/ionization (LDI) method and time of flight (TOF) mass spectrometry are presented. Several liquid fuels (benzene, tetrahydrofuran, toluene, acetonitrile, acetone, isopropanol, ethanol) were used to produce soot samples. Each soot gives specific mass spectrum of carbon clusters C_n as well as their size. The best clustering process was observed for benzene soot with the cluster size n?31. The benzene soot was applied in the detection of insulin by using the matrix-assisted laser desorption/ionization (MALDI) method.     

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.     

Reactive-electrospray-assisted laser desorption/ionization for characterization of peptides and proteins

Electrospray-assisted laser desorption/ionization (ELDI) is a soft ionization method for mass spectrometry (MS) and combines features of both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization to generate ESI-like multiply charged molecules. The ELDI process is based on merging ESI-generated, charged droplets with particles UV laser desorbed from dried or wet sample deposits. We previously reported that ELDI is amenable for MS-based protein identification of large peptides and small proteins using top-down and bottom-up techniques (Peng, I. X.; Shiea, J.; Ogorzalek Loo, R. R.; Loo, J. A. Rapid Commun. Mass Spectrom. 2007, 21, 2541-2546). We have extended our studies by applying collisionally activated dissociation and electron-transfer dissociation MS ( n ) to protein analysis and show that ELDI is capable of multistage MS to MS (4) for top-down characterization of large proteins such as 29 kDa carbonic anhydrase. Multiply charged proteins generated by the ELDI mechanism can be shifted to higher charge by increasing the organic content in the ESI solvent to denature the protein molecules, or by adding m-nitrobenzyl alcohol to the ESI solvent. Furthermore, we introduce "reactive-ELDI", which supports chemical reactions during the ELDI process. Preliminary data for online disulfide bond reduction using dithiothreitol on oxidized glutathione and insulin show reactive-ELDI to be effective. These data provide evidence that the laser-desorbed particles merge with the ESI-generated charge droplets to effect chemical reactions prior to online MS detection. This capability should allow other chemical and enzymatic reactions to be exploited as online protein characterization tools, as well as extending them to flexible, spatially resolved tissue screening and imaging. Also, these reactive-ELDI disulfide reduction experiments enable direct top-down protein identification for proteomic study, side stepping laborious, time-consuming sample preparation steps such as in-solution reduction and alkylation.     

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 saturated hydrocarbons by using chemical ionization combined with laser-induced acoustic desorption/Fourier transform ion cyclotron resonance mass spectrometry

Laser-induced acoustic desorption (LIAD), combined with chemical ionization by the cyclopentadienyl cobalt radical cation (CpCo.+), is demonstrated to facilitate the analysis of saturated hydrocarbons by Fourier transform ion cyclotron resonance mass spectrometry. The LIAD/CpCo.+ method produces unique pseudomolecular ions for alkanes from C(24)H(50) to C(50)H(102). These alkanes were tested individually and in artificial mixtures of up to seven components. Only one product ion, [R + CpCo - 2H(2)].+, was detected for each alkane (R). The product ions' relative abundances correspond to the relative molar concentration of each alkane in mixtures. These findings provide a solid groundwork for the future application of this method for hydrocarbon polymer analyses.     

Direct from polyacrylamide gel infrared laser desorption/ionization

The direct combination of gel electrophoresis and infrared laser desorption/ionization time-of-flight mass spectrometry has been demonstrated. We present results for infrared laser desorption and ionization mass spectrometry of peptides and proteins directly from a polyacrylamide gel without the addition of a matrix. Analyte molecules up to 6 kDa were ionized directly from a vacuum-dried sodium dodecyl sulfate-polyacrylamide gel after electrophoretic separation. Mass spectra were obtained at the wavelength of 2.94 microm, which is consistent with IR absorption by N-H and O-H stretch vibrations of water and other constituents of the gel. A 5-nmol quantity of peptide or protein was loaded per gel slot, although it was possible to obtain mass spectra from a small fraction of the gel spot. This technique shows promise for the direct identification of both parent and fragment masses of proteins contained in polyacrylamide gels.     

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.     

Metal powder substrate-assisted laser desorption/ionization mass spectrometry for polyethylene analysis

Polyethylene is one of the most important industrial polymers and is also one of the most challenging polymers to be characterized by mass spectrometry. We have developed a substrate-assisted laser desorption/ionization (LDI) mass spectrometric method for polyethylene analysis. In this method, cobalt, copper, nickel, or iron metal powders are used as a sample substrate and silver nitrate is used as the cationization reagent. Using a conventional UV LDI time-of-flight mass spectrometer, intact oligomer ions having masses up to 5000 u can be detected. Cobalt is found to produce spectra with the highest signal-to-noise ratio and the lowest level of fragmentation. Cobalt powder size is shown to have some effect on the spectra produced. The best results are obtained with the use of cobalt powders with diameters ranging from 30 to 100 microm. Fragmentation cannot be totally eliminated, but the fragment ion peaks can be readily discerned from the intact polyethylene ions in the substrate-assisted LDI spectrum. Thus, the average molecular masses of low-mass polyethylene samples can be determined by using this method. A rapid heating model is used to account for the effectiveness of using the coarse metal powders to assist the analysis of intact polyethylene molecules by LDI.     

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.     

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.     

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.     

Analysis of polyethylene by using cyclopentadienyl cobalt chemical ionization combined with laser-induced acoustic desorption/fourier transform ion cyclotron resonance mass spectrometry

A new mass spectrometric method has been developed for the analysis of low molecular weight polyethylene (PE). Laser-induced acoustic desorption (LIAD), combined with chemical ionization by the cyclopentadienyl cobalt radical cation (CpCo.+) in a Fourier transform ion cyclotron resonance mass spectrometer, produces predominantly a quasimolecular ion, (R + CpCo - 2H2).+, for each PE oligomer (R). An examination of artificial alkane mixtures revealed no mass bias for alkanes of differing molecular weights. However, the success of the LIAD/CpCo.+ CI technique depends greatly upon the LIAD sample preparation method used. Several sample preparation methods were evaluated, and pneumatically assisted spin coating was concluded to provide the best mass spectra as a result of its ability to provide uniform PE coverage on the LIAD foils. The molecular weight distributions measured for several low molecular weight PE samples (200-655) were found to be in good agreement with manufacturers' values as determined by gel permeation chromatography.     

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.     

Mass spectrometry imaging of small molecules using desorption/ionization on silicon

Development of novel tools to image spatial distribution of small molecules in biological samples is essential in disease diagnosis and biomarker discovery. To simplify sample preparation and reduce background noise in the low-mass region, we describe here the use of a matrix-free mass spectrometric imaging method, i.e., desorption/ionization on silicon (DIOS), for biological surface analysis. The imaging parameters, such as the laser beam diameter and the translation stage movement, were studied and optimized to improve imaging performance. The use of DIOS imaging to map small molecules on mouse liver tissues was demonstrated. In addition, phosphatidylcholine (PC) and propidium iodide (PI) were used as the cell membrane and nucleus markers, respectively, to "visualize" the presence of HEK 293 cells. The reconstructed ion maps of PC and PI were compared with the optical images collected from the same sample using bright-field and fluorescence microscopy. A good correlation of the spatial distribution of cells confirmed the validity of this DIOS imaging approach.