Improving precision in resonance ionization mass spectrometry: influence of laser bandwidth in uranium isotope ratio measurements

The use of broad bandwidth lasers with automated feedback control of wavelength was applied to the measurement of (235)U/(238)U ratios by resonance ionization mass spectrometry (RIMS) to decrease laser-induced isotopic fractionation. By broadening the bandwidth of the first laser in a three-color, three-photon ionization process from a bandwidth of 1.8 GHz to about 10 GHz, the variation in sequential relative isotope abundance measurements decreased from 10% to less than 0.5%. This procedure was demonstrated for the direct interrogation of uranium oxide targets with essentially no sample preparation.     

Determination of iodine in oyster tissue by isotope dilution laser resonance ionization mass spectrometry

The technique of laser resonance ionization mass spectrometry has been combined with isotope dilution analysis to determine iodine in oyster tissue. The long-lived radioisotope, 129I, was used to spike the samples. Samples were equilibrated with the 129I, wet ashed under controlled conditions, and iodine separated by coprecipitation with silver chloride. The analyte was dried as silver ammonium iodide upon a tantalum filament from which iodine was thermally desorbed in the resonance ionization mass spectrometry instrument. A single-color, two-photon resonant plus one-photon ionization scheme was used to form positive iodine ions. Long-lived iodine signals were achieved from 100 ng of iodine. The precision of 127I/129I measurement has been evaluated by replicate determinations of the spike, the spike calibration samples, and the oyster tissue samples and was 1.0%. Measurement precision among samples was 1.9% for the spike calibration and 1.4% for the oyster tissue. The concentration of iodine determined in SRM 1566a, Oyster Tissue, was 4.44 micrograms/g with an estimate of the overall uncertainty for the analysis of +/- 0.12 microgram/g.     

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.     

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.     

High-pressure gas phase femtosecond laser ionization mass spectrometry

We describe a novel ion source for analytical mass spectrometry based on femtosecond laser ionization at pressures at and above atmospheric and characterize its performance when coupled to a tandem quadrupole/time-of-flight mass spectrometer. We assess source saturation limits, ionization and sampling efficiencies, the effective ionization volume, and limits of detection. We demonstrate 100% efficient ionization for a set of organic compounds and show that the degree of ion fragmentation over a range of laser powers is favorable compared to electron impact ionization, especially in that a substantial parent ion signal is always observed. We show how collisional cooling plays a role in controlling fragmentation at high pressures and address how ion-molecule chemistry can be controlled or exploited. High-pressure femtosecond laser ionization will allow "universal" and efficient ionization, presenting a research direction that will broaden the options for gas phase analysis beyond the capabilities of electron impact ionization.     

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.     

Trace uranium analysis by isotope dilution alpha and mass spectrometry and comparison with other techniques

In this paper uranium determination by isotope dilution mass (MSID) and alpha spectrometry (ASID) using ²³³U as an isotope diluent is discussed. In addition, a new form of MSID employing two tracers (²³⁵U and ²³³U) is developed, where two independent uranium values are obtained for single dilution. The precision and accuracy of each technique are evaluated by comparison with other techniques like X-ray fluorescence and instrumental neutron activation analysis. The techniques, employed here for geological samples, can easily be adapted for environmental materials.     

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.     

Elemental imaging via laser ionization orthogonal time-of-flight mass spectrometry

An elemental imaging method using a laser ionization orthogonal time-of-flight mass spectrometer system was developed for the simultaneous detection of all metal and nonmetal elements. The instrument control and data processing were realized by self-developed programs. This system is capable of simultaneous detection of metal and nonmetal elements, with a spatial resolution of 50 μm, the lowest detection limits of 3 × 10(-7) g/g (Li), and a dynamic range of 7 orders of magnitude. Moreover, this technique does not require standards for quantitative analysis and can be a powerful and versatile tool for elemental imaging.     

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.     

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.     

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.     

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.     

Analysis of noncovalent chitinase-chito-oligosaccharide complexes by infrared-matrix assisted laser desorption ionization and nanoelectrospray ionization mass spectrometry

Transferring noncovalently bound complexes from the condensed phase into the gas phase represents a challenging task due to weak intermolecular bonds that have to be maintained during the phase transition. Currently, electrospray ionization (ESI) is the standard mass spectrometric (MS) technique to analyze noncovalent complexes. Although infrared matrix-assisted laser desorption ionization (IR-MALDI)-MS also provides particular soft desorption/ionization conditions, this method has so far hardly been applied for the analysis of noncovalent complexes. In this study, we employed IR-MALDI orthogonal time-of-flight (o-TOF)-MS in combination with the liquid matrix glycerol to characterize the specific complex formation of chito-oligosaccharide (CHOS) ligands with two variants of Chitinase A (ChiA) from Serratia marcescens, the inactive E315Q mutant and the active W167A mutant, respectively. The IR-MALDI-o-TOF-MS results were compared to those obtained using nano-ESI-quadrupole (q)-TOF-MS and ultraviolet (UV)-MALDI-o-TOF-MS. Using IR-MALDI-o-TOF-MS, specific noncovalent complexes between ChiA and CHOS were detected with distributions between enzymes with bound oligosaccharides vs free enzymes that were essentially identical to those obtained by nano-ESI-q-TOF-MS. Chitinase-CHOS complexes were not detected when UV-MALDI was employed for desorption/ionization. The results show that IR-MALDI-MS can be a valuable tool for fast and simple screening of noncovalent enzyme-ligand interactions.     

Portable laser ablation sampling device for elemental fingerprinting of objects outside the laboratory with laser ablation inductively coupled plasma mass spectrometry

Laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) is a powerful method for elemental fingerprinting of solid samples in a quasi-nondestructive manner. In order to extend the field of application to objects outside the laboratory, a portable laser ablation sampling device was assembled using a diode pumped solid state laser and fiber-optics. The ablated materials were sampled on membrane filters and subsequently quantified by means of LA-ICPMS. The analytical performance of this approach was investigated for glass and gold reference materials. Accuracies of better than 20% were reached for most elements and typical limits of detection were found to be in the range of 0.01-1 μg/g. In summary, this approach combines spatially resolved sampling with the detection power of ICPMS and enables elemental fingerprinting of objects which cannot be transferred to the laboratory, e.g., archeological artifacts in museums.     

Direct analysis of single rat peritoneal mast cells with laser vaporization/ionization mass spectrometry

A linear time-of-flight mass spectrometer was used as a detector for flow cytometry. These two techniques were coupled by a laser vaporization/ionization interface. The estimated mass detection limit of the combined system was 20 amol of serotonin standard with one laser pulse. An aqueous buffer at physiological pH was used to ensure compatibility with cells. Rat peritoneal mast cells (RPMCs) were dispensed into the mass spectrometer in a single file confined within a 20-micron-i.d. capillary. By using the mass spectrometer as a detector, no precolumn staining or derivatization is required. Determination of serotonin and histamine in individual cells was demonstrated. With this method, hundreds of cells can be analyzed within a few minutes. The average amounts of histamine and serotonin per RPMC were found to be 0.75 +/- 0.33 and 0.11 +/- 0.06 fmol, respectively. No correlation was found between the amounts of the two amines in each cell.     

Detection of oligosaccharides labeled with cyanine dyes using matrix-assisted laser desorption/ionization mass spectrometry

The sensitivity of oligosaccharides in mass spectrometry lags far behind that of peptides. This is a critical factor in realizing the high-throughput analysis of posttranslational modifications in proteomics. We here described that hydrazide derivatives of cyanine dyes (Cy3, Cy5) with a positive charge made excellent labeling reagents for the detection of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Cy3-labeled standard N-glycan could be detected at 200 amol on the MALDI target plate in reflectron mode without any purification procedures after the labeling reaction, which may meet the level of sensitivity required in proteome research. Despite the general recognition that the production of signals of oligosaccharides under MALDI conditions would be highly dependent on the matrix, most of the known N-glycans from chicken ovalbumin could be detected upon Cye derivatization nearly independent of the kind of matrix tested (e.g., nor-harman, 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid) without spoiling the signal strength. Postsource decay afforded simple spectra mainly consisting of Y-type fragment ions, thus simplifying the sequence analysis. In-source decay afforded a similar fragmentation pattern only when acidic matrixes were used. In addition, this derivatization technique was successfully applied to the profiling of N-glycans of gel-separated glycoproteins.