基质辅助激光解吸电离飞行时间质谱研究2: 四硫 富瓦烯化合物

采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),对四硫富瓦烯化合物进行质谱表征。在所用的实验条件下,样品很容易解吸电离生成单电荷分子离子,得到单同位素分辨的质谱图。26种实际样品的质谱分析结果表明;MALDI-TOF-MS可以比其它质谱方法更有效、更方便地用于此类化合物的质谱分析,解决了此类化合物不易进行质谱鉴定的难题。     

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.     

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.     

Rapid and reliable species identification of wild mushrooms by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)

Mushrooms are a favourite natural food in many countries. However, some wild species cause food poisoning, sometimes lethal, due to misidentification caused by confusing fruiting bodies similar to those of edible species. The morphological inspection of mycelia, spores and fruiting bodies have been traditionally used for the identification of mushrooms. More recently, DNA sequencing analysis has been successfully applied to mushrooms and to many other species. This study focuses on a simpler and more rapid methodology for the identification of wild mushrooms via protein profiling based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). A preliminary study using 6 commercially available cultivated mushrooms suggested that a more reproducible spectrum was obtained from a portion of the cap than from the stem of a fruiting body by the extraction of proteins with a formic acid-acetonitrile mixture (1 + 1). We used 157 wild mushroom-fruiting bodies collected in the centre of Hokkaido from June to November 2014. Sequencing analysis of a portion of the ribosomal RNA gene provided 134 identifications of mushrooms by genus or species, however 23 samples containing 10 unknown species that had lower concordance rate of the nucleotide sequences in a BLAST search (less than 97%) and 13 samples that had unidentifiable poor or mixed sequencing signals remained unknown. MALDI-TOF MS analysis yielded a reproducible spectrum (frequency of matching score ≥ 2.0 was ≥6 spectra from 12 spectra measurements) for 114 of 157 samples. Profiling scores that matched each other within the database gave correct species identification (with scores of ≥2.0) for 110 samples (96%). An in-house prepared database was constructed from 106 independent species, except for overlapping identifications. We used 48 wild mushrooms that were collected in autumn 2015 to validate the in-house database. As a result, 21 mushrooms were identified at the species level with scores ≥2.0 and 5 mushrooms at the genus level with scores ≥1.7, although the signals of 2 mushrooms were insufficient for analysis. The remaining 20 samples were recognized as “unreliable identification” with scores <1.7. Subsequent DNA analysis confirmed that the correct species or genus identifications were achieved by MALDI-TOF MS for the 26 former samples, whereas the 18 mushrooms with poorly matched scores were species that were not included in the database. Thus, the proposed MALDI-TOF MS coupled with our database could be a powerful tool for the rapid and reliable identification of mushrooms; however, continuous updating of the database is necessary to enrich it with more abundant species.     

Au@SiO2 core-shell nanoparticles for laser desorption/ionization time of flight mass spectrometry

In matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), the analysis capability, especially for small molecules, is often compromised by the addition of organic matrices due to the existence of background signals. Herein we report a new detection method on the utility of core-shell nanoparticles (CSNPs) as energy transfer structure in LDI-TOF-MS. The LDI-TOF-MS based on gold-silica core-shell nanoparticles with ultrathin silica shell of 2-4 nm (Au@utSiO(2) CSNPs) was effectively applied to the analysis of many compounds, especially for small functional molecules and polymers, which was more promising than MALDI-TOF-MS.     

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.     

Small molecule matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a polymer matrix

We have employed a light-absorbing electrically conductive polymer as a matrix to determine the molecular mass of small organic molecules using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method, which is in contrast to the usual MALDI strategy for matrix selection in which a small molecule matrix is used with a high molecular mass analyte, addresses the problem of matrix interference which limits the usefulness of MALDI-TOF for small molecule analysis. Use of negative ion mode offers advantages for this application. Using this approach, we have obtained clean molecular ion mass spectra of small organic molecules in the mass range 100-300 Da.     

Small-molecule analysis by surface-assisted laser desorption/ionization mass spectrometry

In order to meet the challenges facing modern chemistry, biology, and medicine, methods are required capable of performing rapid and reliable analysis of both individual compounds and complex mixtures at the molecular level. Matrix-assisted laser de-sorption/ionization mass spectrometry meets these requirements; however, some limitations complicate its application for the analysis of small molecules. Recently, small-molecule analysis has greatly progressed owing to development of surface-assisted laser desorption/ionization mass spectrometry involving approaches which combine the unique properties of nanostructured surface chemistry and morphology. This review examines such approaches and their specific application in small-molecule mass analysis.     

Oligonucleotide analysis by nanoparticle-assisted laser desorption/ionization mass spectrometry

We analyzed oligonucleotides by nanoparticle-assisted laser desorption/ionization (nano-PALDI) mass spectrometry (MS). To this end, we prepared several kinds of nanoparticles (Cr-, Fe-, Mn-, Co-based) and optimized the nano-PALDI MS method to analyze the oligonucleotides. Iron oxide nanoparticles with diammonium hydrogen citrate were found to serve as an effective ionization-assisting reagent in MS. The mass spectra showed both [M - H](-) and [M + xMe(2+)- H](-) (Me: transition metal) peaks. The number of metal-adducted ion signals depended on the length of the oligonucleotide. This phenomenon was only observed using bivalent metal core nanoparticles, not with any other valency metal core nanoparticles. Our pilot study demonstrated that iron oxide nanoparticles could easily ionize samples such as chemical drugs and peptides as well as oligonucleotides without the aid of an oligonucleotide-specific chemical matrix (e.g., 3-hydroxypicolinic acid) used in conventional MS methods. These results suggested that iron-based nanoparticles may serve as the assisting material of ionization for genes and other biomolecules.     

Analysis of triacylglycerols and whole oils by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Since the introduction of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry the majority of research has focused on developing analytical methods for the qualitative determination of water soluble biomolecules such as proteins, peptides, carbohydrates, and oligonucleotides. This paper, however, reports the use of MALDI for the analysis of triacylglycerols and develops a new sample preparation method for nonpolar analytes. MALDI enables the rapid analyses of triacylglycerol (TAG) standards and mixtures of whole oils. The new method provides excellent shot to shot reproducibility, making quantification possible. Detection limits were in the mid femtomole range and the resolution was around 2000 which easily separated TAGs differing by one double bond. Sensitivity decreased with increasing molecular weight, causing biased results when analyzing complex mixtures with a significant range of molecular weight. In all cases only sodiated molecules and prompt losses of a fatty acid sodium salt were observed in the spectra. From this information it was possible to identify the three fatty acids on the glycerol backbone. Collision-induced dissociation was carried out on a triacylglycerol which proved to be useful for additional structural information, including the corroboration of the fatty acid components. With MALDI the percent compositions of TAGs in a standard olive oil was accurately determined. Finally, MALDI was used to examine the differences in lipid components between aged and fresh onion seeds, showing the potential of the technique for observing changes in lipid components in seeds.     

Application of off-line size-exclusion chromatographic fractionation-matrix assisted laser desorption ionization time of flight mass spectrometry for proanthocyanidin characterization

In this work, we wanted to devise a reliable method to characterize polymerized forms of tannins, their structural information and mass distribution. Size-exclusion chromatography (SEC) is a chromatographic technique used to determine molecular mass (weight) distributions of polymers. One important step in the data treatment is the modeling of the calibration curves. Polystyrenes (PS) are standards usually used because no commercial procyanidin (PC) standards are available. An off-line coupling of SEC and MALDI was carried out to measure differences between polystyrenes and procyanidins. Thus, a new calibration curve was established; from 1000 to 8000 Da, there is a good correlation between the MALDI and PS calibration curves, in this field the PS calibration is correct and enables true mass determination. For masses above 8000 Da, PS calibration overestimates the real molecular weight of PC, overestimation of 53%. And for masses below 1000 Da, PS calibration underestimates their real molecular weight (10-15%). This means that to truly characterize PC, calibration based on PC standards is required.     

高效液相色谱-基质辅助激光解吸电离串联飞行时间质谱分离鉴定牛乳铁蛋白素

建立了高效液相色谱-基质辅助激光解吸电离串联飞行时间质谱(MALDI-TOF/TOF MS)分离鉴定牛乳铁蛋白素(bovine lactoferricin, LfcinB)的方法。采用胃蛋白酶酶解牛乳铁蛋白,酶解液离心后取上清液,经过离子交换色谱、反相液相色谱、透析等技术分离,对分离得到的目标产物进行抗菌活性分析、蛋白含量测定和MALDI-TOF/TOF MS鉴定。分离得到高活性的LfcinB,其相对分子质量为3124.89,蛋白含量为18.20 μg/mL。本方法具有精度高、分析速度快和分辨能力强等优点,是其他传统的分析鉴定LfcinB方法所无法比拟的,为进一步研究LfcinB奠定了基础。     

Molecular histology analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry using gold nanoparticles as matrix

Gold nanoparticles (AuNPs) were applied and optimized as matrix for matrix‐assisted laser desorption/ionization mass spectrometry analysis of animal tissues, and enabled histological analysis of animal tissues at molecular level by imaging mass spectrometry (IMS). AuNPs were coated on animal tissue in a solvent‐free manner via argon ion sputtering. Metabolites, including neurotransmitters, fatty acids and nucleobases, were directly detected from mouse brain tissue. Based on region‐specific chemical profiles, fine histological features of mouse brain tissue and heterogeneous regions of tumor tissue were both revealed. Copyright © 2011 John Wiley & Sons, Ltd.     

Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis of hyaluronan oligosaccharides

A new method is presented for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronan (HA) with bacterial hyaluronidase (E.C. 4.2.2.1, from Streptomyces hyalurolyticus) using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS). Mixtures containing HA oligosaccharides of tetrasaccharide (4-mer)-34-mer were analyzed using this method. The carboxyl groups of the glucuronate residues in the prepared HA oligomers, were modified as the acidic form (COOH), sodium salts (COONa), organic ammonium salts, or methylesters before MALDI-TOFMS measurement. Among these samples, the methylester form of glucuronate residues in HA oligosaccharides, prepared by methylation using trimethylsilyl diazomethane, afforded high sensitivity for spectra. This simple modification method for carboxyl group methylation of acidic polysaccharides [Hirano et al., Carbohydr. Res., 340, (2005) 2297-2304] provides samples suitable for MALDI-TOF mass spectrometric analysis throughout a significantly enhanced range of masses.     

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.     

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.