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.     

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.     

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.     

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.     

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.     

Surface cleaning of germanium nanodot ionization substrate for surface‐assisted laser desorption/ionization mass spectrometry

In surface‐assisted laser desorption/ionization mass spectrometry (SALDI‐MS), a chemical background signal, arising from organic contaminants such as plasticizers, is frequently observed mainly under m/z ca. 600, which impairs the advantages of the matrix‐free approach. Silver salts, which are used for the cationization of aromatic compounds, are also difficult to remove completely after the measurements. In this study, surface cleaning techniques used in semiconductor processing were used to clean our developed silicon‐based SALDI substrate on which self‐assembled germanium nanodots (GeNDs) had been deposited (termed a GeND chip). An immersion cleaning method using acetone with sonication, and a sulfuric‐peroxide mixture (SPM) cleaning method using a mixture of H₂SO₄/H₂O₂/deionized water, were examined for their effectiveness in removing organic compounds and residual silver salts. Removal of both types of contaminants was successfully performed by SPM cleaning. The limit of detection for glutathione was improved from ca. 5 pmol without cleaning to ca. 50 fmol after the SPM cleaning. Since GeND chips can tolerate acidic cleaning and sonication due to their chemical inertness and rigid nanodot structures, they appear to be an ideal reusable SALDI substrate. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

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.

Open image in new window

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

     

Immobilization of aminophenylboronic acid on magnetic beads for the direct determination of glycoproteins by matrix assisted laser desorption ionization mass spectrometry

Aminophenylboronic acid (APBA) has been immobilized on magnetic beads for the direct determination of glycoprotein by matrix assisted laser desorption/ionizaton time of flight mass spectrometry (MALDI-TOF-MS). An APBA layer was formed on the surface of carboxylic acid terminated magnetic beads by coupling with carbodiimide and subsequently reacted with an N-hydroxysuccinimide moiety. The immobilized APBA was identified by MALDI-TOF-MS without a matrix. Glycoproteins, such as HbA1c, fibrinogen, or RNase B were separated and desalted using APBA magnetic beads by simply washing the magnetic beads and then separating them by external magnet. Proteins can be identified by direct determination of proteins on beads on MALDI plate and confirmed again by peptide mass finger printing after digestion of proteins on magnetic beads by trypsin. Fluorescence image with a FITC tagging protein using confocal laser microscopy showed the difference of immobilization efficiency between glycoproteins and nonglycoproteins. The methods developed within this work allow the simple treatment and enrichment of glycoproteins as well as direct determination of proteins on beads by MALDI-TOF-MS.     

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.     

Detection of aminothiols through surface‐assisted laser desorption/ionization mass spectrometry using mixed gold nanoparticles

We have employed mixtures of two differently sized (average diameters: 3.5 and 14 nm) gold nanoparticles (Au NPs) as selective probes and matrices for the determination of aminothiols using surface‐assisted laser desorption/ionization mass spectrometry (SALDI‐MS). When using 38 and 150 pM solutions of the 3.5‐ and 14‐nm Au NPs, respectively, as the probe and matrix, SALDI‐MS provided limits of detection (signal‐to‐noise ratio = 3) of 2, 20, and 44 nM for 1.0 mL solutions of glutathione (GSH), cysteine (Cys), and homocysteine, respectively. The signal intensities of these analytes varied by less than 20% for SALDI‐MS analyses recorded over 50 sample spots; in contrast, they varied by as much as 60% when using a conventional matrix (2,5‐dihydroxybenzoic acid). We validated the practicality of this approach – with its advantages of sensitivity, reproducibility, rapidity, and simplicity – through the analysis of GSH in MCF‐7 cell lysates and Cys in plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Capillary isoelectric focusing in pseudo-closed channel coupled to matrix assisted laser desorption/ionization mass spectrometry for protein analysis

Capillary isoelectric focusing (CIEF) was performed in pseudo-closed channel to separate proteins on a plastic chip. Pseudo-closed channel provided a novel way to couple protein separation by CIEF to MALDI mass spectrometry without eluting the focused proteins.     

Matrix-assisted laser desorption ionization mass spectrometry for identification of shrimp

Matrix-assisted laser desorption ionization (MALDI) time of flight mass spectrometry was used to identify shrimp at the species level using commercial mass spectral fingerprint matching software (Bruker Biotyper). In the first step, a mass spectrum reference database was constructed from the analysis of six commercially important shrimp species: Litopenaeus setiferus, Farfantepenaeus aztecus, Sicyonia brevirostris, Pleoticus robustus, Pandalopsis dispar and Pandalus platyceros. This step required a desalting procedure for optimum performance. In the second step, the reference database was tested using 74 unknown shrimp samples from these six species. Correct identification was achieved for 72 of 74 samples (97%): 72 samples were identified at the species level and 2 samples were identified at the genus level using the manufacturer's log score specifications. The MALDI fingerprinting method for the identification of shrimp species was found to be reproducible and accurate with rapid analysis.     

Electrospray-assisted laser desorption/ionization mass spectrometry for direct ambient analysis of solids

A new method of electrospray-assisted laser desorption/ionization (ELDI) mass spectrometry, which combines laser desorption with post-ionization by electrospray, was applied to rapid analysis of solid materials under ambient conditions. Analytes were desorbed from solid metallic and insulating substrata using a pulsed nitrogen laser. Post-ionization produced high-quality mass spectra characteristic of electrospray, including protein multiple charging. For the first time, mass spectra of intact proteins were obtained using laser desorption without adding a matrix. Bovine cytochrome c and an illicit drug containing methaqualone were chosen in this study to demonstrate the applicability of ELDI to the analysis of proteins and synthetic organic compounds.     

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。     

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 the multimeric state of proteins by matrix assisted laser desorption/ionization mass spectrometry after cross-linking with glutaraldehyde

We have undertaken a systematic study on the suitability of matrix-assisted laser desorption/ionization mass spectrometry to analyze and determine the multimericity of several proteins after cross-linking with glutaraldehyde. Using both commercially available proteins and others of viral origin currently being characterized in our laboratory, we studied the range of concentrations of cross-linker and protein for optimal analysis. Under the conditions developed during this study, we confirmed the multimeric states of three phage PRD1 structural proteins with monomeric masses ranging from 13.5 to 63 kDa. In addition, we addressed the question of the general applicability of the method by using it successfully to confirm the stoichiometry of the heptameric chaperonin GroEL, a bacterial protein with a mass well over 450 kDa.     

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.