基质辅助激光解析电离-飞行时间质谱分析寡核苷酸G-四链体

我们发展了一种利用基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF MS)分析对金属离子具有较高亲和力的寡核苷酸G-四链体的方法.考察了不同基质:3-羟基吡啶甲酸(3-HPA)与柠檬酸氢二铵(DHC)混合基质、3,4-二胺基苯基苯甲酮(DABP)及DABP/DHC混合基质,应用于G-四链体分析的效果.实验结果表...     

Analysis of quaternary protein ensembles by matrix assisted laser desorption/ionization mass spectrometry

The intact noncovalent structure of the homo-oligomeric complexes of streptavidin (52 kDa), alcohol dehydrogenase (150 kDa), and beef liver catalase (240 kDa) have been observed using the matrix 2,6-dihydroxyacetophenone in an organic solvent. Intact streptavidin tetramers could also be observed with ferulic acid and other hydroxyacetophenone derivatives. Intact complexes are observed only for the first shot at a given position, which may be due to physical segregation or precipitation of the noncovalent complexes at the crystal surface. This effect is independent of the macroscopic crystal structure or the type of substrate (hydrophobic versus hydrophilic). Observation of intact complexes is not affected by addition of less than 10 mM salts or buffers, and appears to be independent of the pH stability range of the protein samples investigated.     

Nanoparticle assisted laser desorption/ionization mass spectrometry for small molecule analytes

Nanoparticle assisted laser desorption/ionization mass spectrometry (NPs-ALDI-MS) shows remarkable characteristics and has a promising future in terms of real sample analysis. The incorporation of NPs can advance several methods including surface assisted LDI-MS, and surface enhanced LDI-MS. These methods have advanced the detection of many thermally labile and nonvolatile biomolecules. Nanoparticles circumvent the drawbacks of conventional organic matrices for the analysis of small molecules. In most cases, NPs offer a clear background without interfering peaks, absence of fragmentation of thermally labile molecules, and allow the ionization of species with weak noncovalent interactions. Furthermore, an enhancement in sensitivity and selectivity can be achieved. NPs enable straightforward analysis of target species in a complex sample. This review (with 239 refs.) covers the progress made in laser-based mass spectrometry in combination with the use of metallic NPs (such as AuNPs, AgNPs, PtNPs, and PdNPs), NPs consisting of oxides and chalcogenides, silicon-based NPs, carbon-based nanomaterials, quantum dots, and metal-organic frameworks.

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Graphical abstract An overview is given on nanomaterials for use in surface-assisted laser desorption/ionization mass spectrometry of small molecules.

     

Study of synthetic aliphatic copolyamides by time-of-flight matrix assisted laser desorption/ionization mass spectrometry

Synthetic copolyamides based on aliphatic diamines (1,3-propanediamine and 1,4-butanediamine) and dichlorides of aliphatic carboxylic acids (adipic and sebacic acid dichlorides) were investigated using time-of-flight matrix assisted laser desorption/ionization mass spectrometry. Their mass spectra showed peaks for cationized (Na⁺ and K⁺) and protonated (less intense peaks) oligomers with NH₂-NH₂, NH₂-COOH, or COOH-COOH end groups. No cyclic oligomers were detected in the samples. The compositions of oligomers were determined, and the relative reactivities of homologous comonomers in polycondensation were estimated. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1320–1324, July, 2007.     

Charge assisted laser desorption/ionization mass spectrometry of droplets

We propose and evaluate a new mechanism to account for analyte ion signal enhancement in ultraviolet-laser desorption mass spectrometry of droplets in the presence of corona ions. Our new insights are based on timing control of corona ion production, laser desorption, and peptide ion extraction achieved by a novel pulsed corona apparatus. We demonstrate that droplet charging rather than gas-phase ion-neutral reactions is the major contributor to analyte ion generation from an electrically isolated droplet. Implications of the new mechanism, termed charge assisted laser desorption/ionization (CALDI), are discussed and contrasted with those of the laser desorption atmospheric pressure chemical ionization method (LD-APCI). It is also demonstrated that analyte ion generation in CALDI occurs with external electric fields about one order of magnitude lower than those needed for atmospheric pressure matrix assisted laser desorption/ionization or electrospray ionization of droplets.     

Capillary isoelectric focusing coupled offline to matrix assisted laser desorption/ionization mass spectrometry

This work presents several critical details for making cIEF-MALDI-MS a robust technique which will allow for more routine application and aid in automation. This includes emphasis on the hardware necessary for syringe pump mobilization and proper protocol for preventing disruption from gas bubbles. Following these guidelines, excellent elution time reproducibility is demonstrated for six pI markers (RSD <5%). Additionally, the pI markers are used to calibrate the pH gradient and determine experimental pIs of proteins detected offline by mass spectrometry. This was demonstrated using a standard protein mixture of myoglobin and two forms of β-lactoglobulin. Experimental determination of protein pIs and molecular weights were found to be in agreement with literature values. The technical details discussed provide a sound foundation for applying the offline coupling of MALDI-MS with cIEF.     

Quantitative detection of metabolites using matrix-assisted laser desorption/ionization mass spectrometry with 9-aminoacridine as the matrix

Quantitative detection of metabolites is a highly desirable feature in metabolome analyses. Recently, the successful detection of multiple metabolites using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in the negative ion mode employing 9-aminoacridine as the organic matrix was reported (Edwards JL, Kennedy RT. Anal. Chem. 2005; 77: 2201-2209). However, there is little information available on quantitative detection of multiple metabolites using MALDI-MS and in particular the influence changes in metabolite levels have on such detections. We investigated this aspect by spiking a synthetic metabolite cocktail (consisting of 39 metabolites including amino acids, organic acids and phospho-metabolites) with five representative metabolites at increasing concentrations, one metabolite at a time, and assessed the signals from replicate determinations. It was possible to detect quantitative changes in the spiked metabolites. Although analyte suppression was observed, it was possible to observe scenarios where the spiked metabolite had little or no influence on the quantitative detection of some metabolites. It appears that the mass spectral response of the metabolite is suppressed only when the spiked chemical species are relatively similar in chemical terms. This suggests that quantitation is possible in scenarios where changes in a specific metabolite or a class of metabolites are monitored following appropriate analyte separation strategies, and that careful interpretations must be made when using the technique for quantitative analysis in unbiased metabolomic approaches.     

Rapid drug detection in oral samples by porous silicon assisted laser desorption/ionization mass spectrometry

The demand for analysis of oral fluid for illicit drugs has arisen with the increased adoption of roadside testing, particularly in countries where changes in legislation allow random roadside testing of drivers for the presence of a palette of illicit drugs such as methamphetamine (MA), 3,4‐methylenedioxymethamphetamine (MDMA) and Δ⁹‐tetrahydrocannabinol (THC). Oral samples are currently tested for such drugs at the roadside using an immunoassay‐based commercial test kit. Positive roadside tests are sent for confirmatory laboratory analysis, traditionally by means of gas chromatography/mass spectrometry (GC/MS). We present here an alternative rapid analysis technique, porous silicon assisted laser desorption/ionization time‐of‐flight mass spectrometry (pSi LDI‐MS), for the high‐throughput analysis of oral fluids. This technique alleviates the need for sample derivatization, requires only sub‐microliter sample volumes and allows fast analysis (of the order of seconds). In this study, the application of the technique is demonstrated with real samples from actual roadside testing. The analysis of oral samples resulted in detection of MA and MDMA with no extraction and analysis of THC after ethyl acetate extraction. We propose that, subject to miniaturization of a suitable mass spectrometer, this technique is well suited to underpin the deployment of oral fluid testing in the clinic, workplace and on the roadside. Copyright © 2009 John Wiley & Sons, Ltd.     

Selective detection of homocysteine by laser desorption/ionization mass spectrometry

This article describes the use of 2,3-naphthalenedicarboxaldehyde (NDA) as a selective probe for the determination of homocysteine (HCys) via fluorescence measurement and laser desorption/ionization mass spectrometry (LDI-MS). The derivatives of three aminothiols-HCys, glutathione (GSH), and gamma-glutamylcysteine (gamma-Glu-Cys)-with NDA under alkaline conditions possess different fluorescence emission characteristics, which allow us to identify them from amines, amino acids, and thiols. By selecting appropriate pH and excitation wavelengths, the limits of detection (LODs) at a signal-to-noise ratio of 3 were 5.2, 1.4 and 16 nM for HCys, GSH and gamma-Glu-Cys, respectively. Additionally, strong UV absorption of the NDA-HCys derivative was further observed at 331 nm; it could be directly detected by LDI-MS with a 337-nm nitrogen laser. Selective detection of HCys has been achieved by conducting the LDI-MS of the NDA-HCys derivative, which was found at m/z 406.9. The lowest detectable concentration of the NDA-HCys derivative in this approach was 500 nM. Quantitative determination of HCys in urine samples was accomplished by LDI-MS. Also, a calibration curve was created from plasma samples spiked with standard HCys (20-100 microM). The experimental results suggest that our proposed methods have great potential in clinical diagnosis and metabolomics application.     

A binary matrix for improved detection of phosphopeptides in matrix‐assisted laser desorption/ionization mass spectrometry

Application of matrix‐assisted laser‐desorption/ionization mass spectrometry (MALDI MS) to analysis and characterization of phosphopeptides in peptide mixtures may have a limitation, because of the lower ionizing efficiency of phosphopeptides than nonphosphorylated peptides in MALDI MS. In this work, a binary matrix that consists of two conventional matrices of 3‐hydroxypicolinic acid (3‐HPA) and α‐cyano‐4‐hydroxycinnamic acid (CCA) was tested for phosphopeptide analysis. 3‐HPA and CCA were found to be hot matrices, and 3‐HPA not as good as CCA and 2,5‐dihydroxybenzoic acid (DHB) for peptide analysis. However, the presence of 3‐HPA in the CCA solution with a volume ratio of 1:1 could significantly enhance ion signals for phosphopeptides in both positive‐ion and negative‐ion detection modes compared with the use of pure CCA or DHB, the most common phosphopeptide matrices. Higher signal intensities of phosphopeptides could be obtained with lower laser power using the binary matrix. Neutral loss of the phosphate group (?80 Da) and phosphoric acid (?98 Da) from the phosphorylated‐residue‐containing peptide ions with the binary matrix was decreased compared with CCA alone. In addition, since the crystal shape prepared with the binary matrix was more homogeneous than that prepared with DHB, searching for ‘sweet’ spots can be avoided. The sensitivity to detect singly or doubly phosphorylated peptides in peptide mixtures was higher than that obtained with pure CCA and as good as that obtained using DHB. We also used the binary matrix to detect the in‐solution tryptic digest of the crude casein extracted from commercially available low fat milk sample, and found six phosphopeptides to match the digestion products of casein, based on mass‐to‐charge values and LIFT TOF‐TOF spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

Carbon nanotubes as adsorbent of solid-phase extraction and matrix for laser desorption/ionization mass spectrometry

A method with carbon nanotubes functioning both as the adsorbent of solid-phase extraction (SPE) and the matrix for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) to analyze small molecules in solution has been developed. In this method, 10 microL suspensions of carbon nanotubes in 50% (vol/vol) methanol were added to the sample solution to extract analytes onto surface of carbon nanotubes because of their dramatic hydrophobicity. Carbon nanotubes in solution are deposited onto the bottom of tube with centrifugation. After removing the supernatant fluid, carbon nanotubes are suspended again with dispersant and pipetted directly onto the sample target of the MALDI-MS to perform a mass spectrometric analysis. It was demonstrated by analysis of a variety of small molecules that the resolution of peaks and the efficiency of desorption/ionization on the carbon nanotubes are better than those on the activated carbon. It is found that with the addition of glycerol and sucrose to the dispersant, the intensity, the ratio of signal to noise (S/N), and the resolution of peaks for analytes by mass spectrometry increased greatly. Compared with the previously reported method by depositing sample solution onto thin layer of carbon nanotubes, it is observed that the detection limit for analytes can be enhanced about 10 to 100 times due to solid-phase extraction of analytes in solution by carbon nanotubes. An acceptable result of simultaneously quantitative analysis of three analytes in solution has been achieved. The application in determining drugs spiked into urine has also been realized.     

Solid phase microextraction with matrix assisted laser desorption/ionization introduction to mass spectrometry and ion mobility spectrometry

Solid phase microextraction (SPME) with matrix assisted laser desorption/ionization (MALDI) introduction was coupled to mass spectrometry and ion mobility spectrometry. Nicotine and myoglobin in matrix 2,5-dihydroxybenzonic acid (DHB), enkephalin and substance P in alpha-cyano-4-hydroxy cinnaminic acid were investigated as the target compounds. The tip of an optical fiber was silanized for extraction of the analytes of interest from solution. The optical fiber thus served as the sample extraction surface, the support for the sample plus matrix, and the optical pipe to transfer the laser energy from the laser to the sample. The MALDI worked under atmospheric pressure, and both an ion mobility spectrometer and a quadrupole/time-of-flight mass spectrometer were used for the detection of the SPME/MALDI signal. The spectra obtained demonstrate the feasibility of the SPME with MALDI introduction to mass spectrometry instrumentation.     

Quantitative analysis of technical polymer mixtures by matrix assisted laser desorption/ionization time of flight mass spectrometry

We report quantitative MALDI-TOF measurements for polydimethylsiloxane (PDMS) of two different molecular weights using the relative ratio of the signal intensities of integrated oligomer distributions for these two molecular weight distributions. By reporting the ratio of intensities of the integrals of two oligomer distributions, we assume that the ionization and desorption efficiencies, crystallization conditions and other factors affecting intensity are similar. Poly(methyl methacrylate) (PMMA-33,000) was mixed with PDMS samples to show whether the presence of another material might affect the desorption efficiency. Quantitative values for the number-average molecular weight (Mn), weight-average molecular weight (Mw) and polydispersities (D) were calculated using the oligomer distributions. The results show a linear relationship between the analyte concentrations and the signal intensities in the range from 1,000 Da to 10,000 Da, and the desorption efficiency of these two PDMS materials was the same even in the presence of PMMA.     

Surfactant-mediated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of small molecules

A variety of surfactants have been tested as matrix-ion suppressors for the analysis of small molecules by matrix-assisted laser desorption/ionization time-of flight mass spectrometry. Their addition to the common matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) greatly reduces the presence of matrix-related ions when added at the appropriate mole ratio of CHCA/surfactant, while still allowing the analyte signal to be observed. A range of cationic quaternary ammonium surfactants, as well as a neutral and anionic surfactant, was tested for the analysis of phenolics, phenolic acids, peptides and caffeine. It was found that the cationic surfactants, particularly cetyltrimethylammonium bromide (CTAB), were suitable for the analysis of acidic analytes. The anionic surfactant, sodium dodecyl sulfate, showed promise for peptide analysis. For trialanine, the detection limit was observed to be in the 100 femtomole range. The final matrix/surfactant mole ratio was a critical parameter for matrix ion suppression and resulting intensity of analyte signal. It was also found that the mass resolution of analytes was improved by 25-75%. Depth profiling of sample spots, by varying the number of laser shots, revealed that the surfactants tend to migrate toward the top of the droplet during crystallization, and that it is likely that the analyte is also enriched in this surface region. Here, higher analyte/surfactant concentration would reduce matrix-matrix interactions (known to be a source of matrix-derived ions).     

High-sensitivity matrix-assisted laser desorption/ionization Fourier transform mass spectrometry analyses of small carbohydrates and amino acids using oxidized carbon nanotubes prepared by chemical vapor deposition as matrix

In matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) analyses of small oligosaccharides and amino acids, high sensitivities for oligosaccharides (10 fmol) were obtained by introducing oxidized carbon nanotubes (CNTs) with short and open-end structure as valuable matrix. The CNTs were deposited in porous anodic alumina (PAA) templates by chemical vapor deposition. Transmission electron microscopy (TEM) images show that those CNTs include low levels of amorphous carbon. Thus, the background interference signals generally caused by amorphous carbon powder in CNTs can be reduced effectively. Experiments also confirmed that the FTMS signal intensity of CNTs prepared in PAA template is much lower than that of commercial multi-wall carbon nanotubes (MCNTs). Moreover, the purified process for CNTs with mixed acid (H₂SO₄ and HNO₃) also contributed to the minimization of background. Intense signals corresponding to alkali cation adduct of neutral carbohydrates and amino acids have been acquired. In addition, reliable quantitative analyses for urine and corn root were also achieved successfully. The present work will open a new way to the application of oxidized CNTs as an effective matrix in MALDI MS research.     

Oxidized carbon nanotubes as matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of biomolecules

Oxidized carbon nanotubes (CNTs), which can form a stable homogeneous suspension in water close to a solution phase, were synthesized and used for matrix-assisted desorption/ionization mass spectrometric (MALDI-MS) analysis of biomolecules. Infrared (IR) spectra, transmission electron microscopy (TEM) and particle size analysis were used for the characterization of the oxidized CNTs. The results indicate that the physical structure of the CNTs was not damaged, but carboxylate groups were introduced onto the surface of the CNTs. In addition, impurities including amorphous carbon, which is one of the main reasons for ion source contamination, were destroyed by the oxidization. The carboxyl groups on the oxidized surface of the CNTs can not only provide an additional proton source, but can also increase the surface polarity and solubility of the CNTs, making it easier to manipulate which is important for MALDI analysis of some biomolecules, especially larger peptides and proteins. The oxidized CNTs were successfully applied to the analysis of neutral oligosaccharides, peptides, and insulin, and thus promise to be an efficient matrix for MALDI-MS analysis of biomolecules.     

Detection of water-soluble vitamins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using porphyrin matrices

The detection of water-soluble vitamins B(1), B(2), B(6), B(12) and C by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) was attempted by studying 17 porphyrin matrices. Comparative studies of porphyrin matrices, useful mass spectral window, matrix/analyte molar ratio (M/A), ultraviolet-visible absorption characteristics and quantitative results were made. Most porphyrin matrices provide a useful mass spectral window in the low-mass range. The optimal M/A increases with increasing molecular mass of the vitamin. Vitamin B(12) possesses the highest molecular mass and requires a higher M/A. The presence of hydroxyl or carboxyl groups in the porphyrin is an indicator of a useful MALDI matrix. Vitamins B(2) and B(6) were readily ionized upon irradiation with a 337 nm laser without the use of any porphyrin matrix. Improved linearity and sensitivity of the calibration curves were obtained with samples prepared with a constant M/A. The limits of detection and quantitation are at the picomole level. The results indicate that MALDI-TOFMS with porphyrin matrices is a rapid and viable technique for the detection of low molecular mass water-soluble vitamins.     

Microwave-assisted synthesis and electrochemical capacitance of polyaniline/multi-wall carbon nanotubes composite

Polyaniline/multi-wall carbon nanotubes composite (PANI/MWNTs) was rapidly synthesized by microwave-assisted polymerization. Transmission electron microscope (TEM) image revealed that this composite was a core–shell structure with PANI layers (50–70 nm). Electrochemical behavior of the composite was evaluated by cyclic voltammetry (CV) and galvanostatic charge–discharge tests with a two-electrode system. An enhanced specific capacitance of 322 F/g with a specific energy density of 22 W h/kg was about 12 times that of MWNTs. This composite also exhibited a good rate capability, retaining up to 87% of initial capacity at a current density of 5 mA/cm².