A metabolomic protocol for plant systematics by matrix-assisted laser-desorption/ionization time-of flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) has been widely used for the identification and classification of microorganisms based on their proteomic fingerprints. However, the use of MALDI-TOF MS in plant research has been very limited. In the present study, a first protocol is proposed for metabolic fingerprinting by MALDI-TOF MS using three different MALDI matrices with subsequent multivariate data analysis by in-house algorithms implemented in the R environment for the taxonomic classification of plants from different genera, families and orders. By merging the data acquired with different matrices, different ionization modes and using careful algorithms and parameter selection, we demonstrate that a close taxonomic classification can be achieved based on plant metabolic fingerprints, with 92% similarity to the taxonomic classifications found in literature. The present work therefore highlights the great potential of applying MALDI-TOF MS for the taxonomic classification of plants and, furthermore, provides a preliminary foundation for future research.     

Rapid identification of microorganisms by mass spectrometry: improved performance by incorporation of in-house spectral data into a commercial database

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used as a microbial diagnostic method for species identification of pathogens. However, MALDI-TOF identification of bacteria at the species level remains unsatisfactory, with the major problem being an incomplete database that still needs refinement and expansion. Augmentation of the original MALDI BioTyper 2.0 (Bruker) database by incorporating mass spectra obtained in-house from clinical isolates may increase the identification rate at the species level. We conducted a prospective study to assess whether the augmented database can improve the performance of MALDI-TOF MS for routine identification of species. Cluster analyses revealed distinct differences in MS spectral profiles of clinical isolates obtained in our hospital and those of ATCC strains in the Bruker database. In the first part of the study, which was performed over 3 weeks, 259 bacterial isolates were subjected to analysis by MALDI-TOF MS, and MS spectra of 229 successfully identified isolates (49 species) were incorporated into the original database to give the augmented Bruker-Chiba database. In a second separate analysis, the concordance of identification of 498 clinical isolates of the 49 species with conventional methods was 87.1% (434/498) with the commercial Bruker database and 98.0% (488/498) using the Bruker-Chiba database. These results indicate that refinement of a commercial database can be achieved relatively easy and effectively by incorporating MS spectra of clinical isolates obtained in a clinical laboratory.     

Recent studies on advance spectroscopic techniques for the identification of microorganisms: A review

The continuous development of resistance to antibiotic drugs by microorganisms causes high mortality and morbidity. Pathogens with distinct features and biochemical abilities make them destructive to human health. Therefore, early identification of the pathogen is of substantial importance for quick ailments and healthcare outcomes. Several phenotype methods are used for the identification and resistance determination but most of the conventional procedures are time-consuming, costly, and give qualitative results. Recently, great focus has been made on the utilization of advanced techniques for microbial identification. This review is focused on the research studies performed in the last five years for the identification of microorganisms particularly, bacteria using advanced spectroscopic techniques including mass spectrometry (MS), infrared (IR) spectroscopy, Raman spectroscopy (RS), and nuclear magnetic resonance (NMR) spectroscopy. Among all the techniques, MS techniques, particularly MALDI-TOF/MS have been widely utilized for microbial identification. A total of 44 bacteria i.e., 6 Staphylococcus spp., 3 Enterococcus spp., 6 Bacillus spp., 4 Streptococcus spp., 6 Salmonella spp., and one from each genus including Escherichia, Acinetobacter, Pseudomonas, Proteus, Clostridioides, Candida, Brucella, Burkholderia, Francisella, Yersinia, Moraxella, Vibrio, Shigella, Serratia, Citrobacter, and Haemophilus (spp.) were discussed in the review for their identification using the above-mentioned techniques. Among all the identified microorganisms, 21% of studies have been conducted for the identification of E. coli, 14% for S. aureus followed by 37% for other microorganisms.     

Liquid chromatography/tandem mass spectrometry of unusual phenols from Yucca schidigera bark: comparison with other analytical techniques

Qualitative and quantitative analyses of phenolic compounds are of interest for both medicinal and food plants. In the present work, the phenolic fraction from Yucca schidigera, a plant bearing the GRAS (Generally Recognized as Safe) label approved by the US Food and Drug Administration, was studied. Crude extracts of Y. schidigera bark were investigated by liquid chromatography/UV spectrophotometry with diode-array detection, liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), in order to develop and optimize simple and rapid techniques to determine both stilbenes and yuccaols for the purposes of quality control of collected material. With optimal LC and MS conditions, stilbenes and yuccaols were quantified with all the proposed methods and the results were compared. Sensitivity was evaluated and the results indicated that MS/MS detection in the multiple reaction monitoring mode is easily applicable to this plant and allows the rapid and direct identification and quantification of these peculiar compounds in crude plant extracts.     

Discrimination of Penicillium isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral 'fingerprints' for twelve Penicillium species. Prior to MALDI-TOF MS analysis, eight replicate cultures of each Penicillium species were subjected to three one-minute bead-beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contained abundant peaks in the range of m/z 5000-20 000, and allowed unambiguous discrimination between species. In addition, a biomarker common to all Penicillium mass spectra was observed at m/z 13 900. Discriminant analysis using the MALDI-TOF MS data yielded classification error rates of 0% (i.e. 100% correct identification), indicating that MALDI-TOF MS data may be a useful diagnostic tool for the objective identification of Penicillium species of environmental and clinical importance.     

Automated protein identification using atmospheric-pressure matrix-assisted laser desorption/ionization

Atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) ion trap mass spectrometry (ITMS) has been evaluated for automated protein identification. By using signal averaging and long ion-injection times, protein identification limits in the 50-fmol range are achieved for standard protein digests. Data acquisition requires 7.5 min or less per sample and the MS/MS spectra files are automatically processed using the SEQUEST database searching algorithm. AP-MALDI-ITMS was compared with the widely used methods of microLC/MS/MS (ion trap) and automated MALDI-TOF peptide mass mapping. Sample throughput is 10-fold greater using AP-MALDI compared with microcapillary liquid chromatography/tandem mass spectrometry (microLC/MS/MS). The protein sequence coverage obtained from AP-MALDI-MS/MS spectra matched by SEQUEST is lower compared with microLC/MS/MS and MALDI-TOF mass mapping. However, by using the AP-MALDI full-scan peptide mass fingerprint spectrum, sequence coverage is increased. AP-MALDI-ITMS was applied for the analysis of Coomassie blue stained gels and was found to be a useful platform for rapid protein identification.     

SpectraBank: an open access tool for rapid microbial identification by MALDI-TOF MS fingerprinting

MALDI-TOF MS has proved to be an accurate, rapid, and cost-effective technique for microbial identification in which the spectral fingerprint of an unknown strain can be compared to a database of spectra from reference strains. Most of the existing databases are private and often costly to access, and little spectral information is shared among researchers. The objective of the present communication is to introduce the SpectraBank database (http://www.spectrabank.org), which provides open access MALDI-TOF mass spectra from a variety of microorganisms. This work aims to familiarize readers with the SpectraBank database, from the sample preparation, data collection, and data analysis to how the spectral reference data can be used for microbial species identification. The database currently includes more than 200 MALDI-TOF MS spectra from more than 70 bacterial species and links to the freely available web-based application SPECLUST (http://bioinfo.thep.lu.se/speclust.html) to allow comparisons of the obtained peak mass lists and evaluate phyloproteomic relationships. The SpectraBank database is intended to be expanded by the addition of new spectra from microbial strains, obtained in our laboratory and by other researchers.     

Direct antigen detection from immunoprecipitated beads using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; a new method for immunobeads-mass spectrometry (iMS)

One-step detection of biological molecules is one of the principal techniques for clinical diagnosis, and the potential of mass spectrometry for biomarker detection has been a promising new approach in the field of medical sciences. We demonstrate here a new and high-sensitivity method that we termed immunobeads-mass spectrometry (iMS), which combines conventional immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The key feature of iMS is the MS-compatible condition of immunoprecipitation using detergents with a monosaccaride-C8 alkyl chain or a disaccharide-C10 alkyl chain, and the minimized number of steps required for high-sensitivity detection of target peptides in serum or biological fluid. This was achieved by optimizing the wash buffer and subjecting the immunobeads directly to MALDI-TOF MS analysis. Using this method, we showed that 1 fmol of amyloid beta peptide spiked in serum was readily detectable, demonstrating the powerful tool of iMS as a biomarker detection method.     

Integration of ion-exchange chromatography fractionation with reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for isolation and identification of compounds in Psoralea corylifolia

An approach for the separation and identification of components in a traditional Chinese medicine Psoralea corylifolia was developed. Ion-exchange chromatography (IEC) was applied for the fractionation of P. corylifolia extract, and then followed by concentration of all the fractions with rotary vacuum evaporator. Each of the enriched fractions was then further separated on an ODS column with detection of UV absorbance and atmospheric pressure chemical ionization mass spectrometer (APCI/MS), respectively, and also analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) with matrix of oxidized carbon nanotubes. Totally more than 188 components in P. corylifolia extract were detected with this integrated approach, and 12 of them were preliminary identified according to their UV spectra and mass spectra performed by APCI/MS and MALDI-TOF/MS. The obtained analytical results not only demonstrated the powerful resolution of integration IEC fractionation with reversed-phase liquid chromatography (RPLC)-APCI/MS and MALDI-TOF/MS for analysis of compounds in a complex sample, but also exhibited the superiority of APCI/MS and MALDI-TOF/MS for identification of low-mass compounds, such as for study of traditional Chinese medicines (TCMs) and metabolome.     

Identification of proteins separated by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis with matrix-assisted laser desorption/ionization ion trap mass spectrometry; comparison with matrix-assisted laser desorption/ionization time-of-flight mass fingerprinting

Digests from ten gel bands containing low abundance proteins were analyzed by both matrix-assisted laser desorption/ionization ion trap (MALDI-IT) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) methods. MALDI-TOF techniques were able to identify only one protein from all 10 gel bands, while MALDI-IT identified eight proteins from the same 10 bands. The ability to perform MS/MS experiments with a MALDI-IT instrument leads to protein identifications based on both peptide molecular mass and sequence information, and is much less prone to errors and uncertainties introduced by peptide fingerprinting methodologies in which protein identification is based on peptide molecular masses alone.     

Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis

Current analytical techniques in protein identification by mass spectrometry are based on the generation of peptide mass maps or sequence tags that are idiotypic for the protein sequence. This work reports on the development of the use of mass spectrometric methods for protein identification in research on metabolic pathways of a genetically modified strain of the baker's yeast Saccharomyces cerevisiae. This study describes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass mapping and liquid chromatography/quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) sequence tag analysis in identification of yeast proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The spots were selected for analysis in order to collect information for future studies, to cover the whole pI range from 3 to 10, and to evaluate information from spots of different intensities. Mass mapping as a rapid, high-throughput method was in most cases sensitive enough for identification. LC/MS/MS was found to be more sensitive and to provide more accurate data, and was very useful when analyzing small amounts of sample. Even one sequence tag acquired by this method could be enough for unambiguous identification, and, in the present case, successfully identified a point mutation.     

利用MALDI-TOF MS鉴定芽苗菜中解鸟氨酸拉乌尔菌和肺炎克雷伯菌的研究

应用基质解析电离飞行时间质谱(MALDI-TOF MS)方法对芽苗菜中检出率较高的2种微生物进行鉴定,结果为解鸟氨酸拉乌尔菌(33.3%)和肺炎克雷伯菌(43.5%)。应用SARAMIS Premium软件对这2种微生物的蛋白质图谱进行分析,获得解鸟氨酸拉乌尔菌特征标识峰16组,肺炎克雷伯菌特征标识峰10组;对分离微生物进行全标识峰和加权特征峰相关性分析,结果表明特征峰的加权会影响微生物鉴定结果间的相关关系,增强微生物的辨识度;对两种微生物进行多方面比较,发现两种微生物生化特性比较接近,但16Sr DNA分辨率较高,质谱相关分析虽有相交但可辨识。实验结果表明MALDI-TOF MS具有检测芽苗菜中微生物的能力并在其特征分析及溯源研究上具有一定的优势,MALDI-TOF MS为开放平台,对农产品微生物蛋白质特征图谱的收集与优化可提高其鉴定准确性。     

Analysis of complex phthalic acid based polyesters by the combination of size exclusion chromatography and matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used in conjunction with size exclusion chromatography (SEC) to investigate a model polyester system based on phthalic anhydride–1,2-propylene glycol. The polyesters were synthesized with a 30% molar excess of glycol, with kinetic samples being removed during different intervals of the polyesterification reaction. SEC was used to track the course of the reaction by determining the molecular weight and molecular weight distributions before subsequent off-line coupling with MALDI-TOF MS as a selective detection method to determine the chemical composition, identify the functionality type distributions as well as assist in assigning structural conformations. Mass spectrometry analysis proved to be a highly effective tool to facilitate the identification of the narrowly dispersed fractions obtained from the chromatographic separations as well as serve as a core method to investigate the heterogeneous nature of the bulk kinetic samples. Through the hyphenation of these sophisticated polymer characterization techniques, information on the molecular heterogeneity of the model polyesters, showing a complex variety of possible distributions, was obtained.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry monitoring of anthocyanins in extracts from Arabidopsis thaliana leaves

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.     

Differently complex oligosaccharides can be easily identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry directly from a standard thin-layer chromatography plate

Thin-layer chromatography (TLC) is a simple, fast and inexpensive separation method. Unambiguous identification of the TLC spots is, however, often a problem. Here we show for the first time that oligosaccharides (derived from dextran, alginate, hyaluronan and chondroitin sulfate) can be characterized by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) directly on a TLC plate. The applied oligosaccharides were either commercially available or obtained from the polysaccharides by HCl-induced hydrolysis. Normal phase TLC was followed by MALDI-TOF MS subsequent to matrix deposition. It will be shown that high quality mass spectra can be obtained that enable unequivocal assignments. It will also be shown that the high content of formic acid in the solvent system does not confer major problems but is responsible for the partial formylation of the analyte and minor N-acetyl loss from hyaluronan and chondroitin sulfate.     

Discrimination between wild-type and ampicillin-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was optimized to discriminate between wild-type and ampicillin-resistant Escherichia coli. Only ampicillin-resistant E. coli displayed an m/z ≈ 29,000 peak, which was confirmed as β-lactamase by in-gel digestion followed by peptide mass fingerprinting. Rapid MALDI-TOF MS detection of antibiotic-resistance could fulfill an important clinical need, providing critical phenotypic information beyond genus–species identification.     

Application of thin‐layer chromatography/infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry to structural analysis of bacteria‐binding glycosphingolipids selected by affinity detection

Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin‐layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry (IR‐MALDI‐o‐TOF‐MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL‐specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria‐specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time‐consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo‐series neutral GSLs recognized by P‐fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR‐MALDI‐o‐TOF‐MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate‐specific pathogens involved in human infectious diseases. Copyright © 2010 John Wiley & Sons, Ltd.     

DNA assay based on monolayer-barcoded nanoparticles for mass spectrometry in combination with magnetic microprobes

Mass spectrometry (MS) based methodology offers simple, fast and sensitive diagnosis. While it has become the predominate approach in biomolecular analysis, it has not been suitable for analyzing nucleic acid due to its low ionization efficiency. We report herein on a DNA assay based on monolayer-barcoded nanoparticles that were encoded with reporter mass molecules, which act as surrogate molecules for the matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS) identification of target DNA through mass spectrometry in combination with magnetic microprobes. This assay demonstrated high MS sensitivity, with the ability to detect target DNA at femtomolar (10⁻¹⁵ M) levels. This inaugural effort using combined techniques is significant because it showed an extraordinary analytical capability for differentiating the single nucleotide polymorphism (SNP), which comprises the most abundant source of genetic variation in the human genome. We also report herein the feasibility of MS detection of two target DNAs that have the same mass but different nucleotide base composition, which classic MS methodology is inherently unable to differentiate.     

MALDI-TOF MS analysis of native and permethylated or benzimidazole-derivatized polysaccharides

MALDI-TOF MS provides rapid and sensitive analyses of larger biomolecules. However, MS analyses of polysaccharide have been reported to have lower sensitivity compared to peptides and proteins. Here, we investigated some polysaccharides chemically derivatized by permethylation and ortho-phenylene diamine (OPD) tagging. Methylated glycan is obviously able to improve the sensitivity for mass spectrometry detection. Oxidative condensation by UV-activation tagging to saccharides by OPD and peptide-OPD also improve the sensitivity of MALDI-TOF MS analyses. Polysaccharides including dextran, glucomannan, arabinoxylan, arabinogalactan and beta-1,3-glucan, isolated from nutritional supplements of Ganoderma lucidum and Saccharomyces pastorianus were measured using MALDI-TOF MS with 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix. These glycans were also derivatized to methylated and benzimidazole-tagged glycans by chemical transformation for molecular weight analysis. The derivatized polysaccharides showed excellent MALDI-TOF MS signal enhancement in the molecular weight range from 1 to 5 kDa. Here, we demonstrate an efficient method to give glycan-benzimidazole (glycan-BIM) derivatives for polysaccharide determination in MALDI-TOF MS. Therefore, permethylated or benzimidazole-derivatized polysaccharides provide a new option for polysaccharide analysis using MALDI-TOF MS.     

Mass spectrometry in biodefense

Potential agents for biological attacks include both microorganisms and toxins. In mass spectrometry (MS), rapid identification of potential bioagents is achieved by detecting the masses of unique biomarkers, correlated to each agent. Currently, proteins are the most reliable biomarkers for detection and characterization of both microorganisms and toxins, and MS-based proteomics is particularly well suited for biodefense applications. Confident identification of an organism can be achieved by top-down proteomics following identification of individual protein biomarkers from their tandem mass spectra. In bottom-up proteomics, rapid digestion of intact protein biomarkers is again followed by MS/MS to provide unambiguous bioagent identification and characterization. Bioinformatics obviates the need for culturing and rigorous control of experimental variables to create and use MS fingerprint libraries for various classes of bioweapons. For specific applications, MS methods, instruments and algorithms have also been developed for identification based on biomarkers other than proteins and peptides.