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Most of the clinical isolates, identified as P. putida or P. fluorescens , were misidentified in clinical laboratories. Whole-genome sequencing (WGS) revealed that these isolates belonged to different Pseudomonas species, including novel species. WGS is a gold-standard method to identify Pseudomonas species, and MALDI-TOF MS analysis has the potential to complement WGS to reliably identify them.

Cutibacterium acnes is a major commensal human skin bacteria. It is a producer of propionic acids that maintain skin acidic pH to inhibit the growth of pathogens. On the other hand, it is also associated with diseases such as acne vulgaris and sarcoidosis. C. acnes strains have been classified into six phylotypes using DNA-based approaches. Because several characteristic features of C. acnes vary according to the phylotype, the development of a practical method to identify these phylotypes is needed. For rapid identification of phylotypes for C. acnes strains, a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) fingerprinting technique has been applied; however, some phylotypes have not been discriminated. We developed a high-throughput protein purification method to detect biomarker proteins by ultrafiltration. MALDI-MS proteotyping using profiling of identified biomarker peaks was applied for the classification of 24 strains of C. acnes, and these were successfully classified into the correct phylotypes. This is a promising method that allows the discrimination of C. acnes phylotypes independent of a DNA-based approach.

A Gram-negative, aerobic, rod-shaped, non-spore-forming bacterium, designated as strain BML3T, was isolated from a sputum sample of a hospital patient in Japan. Strain BML3T grew at temperatures from 4 to 40 °C, in 1.0–7.0 % (w/v) NaCl and at pH 6.0–9.0. Results of phylogenetic analysis based on the sequences of housekeeping genes, including the 16S rRNA gene and rpoB, rpoD and gyrB, showed that strain BML3T was part of the Pseudomonas putida group and located close to Pseudomonas asiatica , Pseudomonas monteilii and P. putida . Whole-genome comparisons, using average nucleotide identity and digital DNA–DNA hybridization, confirmed strain BML3T to be a distinct species among the P. putida group. Phenotypic characterization tests demonstrated that the utilization of phenylmercuric acetate could distinguish this strain from other closed species of the P. putida group. Based on genetic and phenotypic evidence, strain BML3T should be classified as a novel species, for which the name Pseudomonas juntendi sp. nov. is proposed. The type strain is BML3T (=DSM 109244T,=JCM 33395T), with a DNA G+C content of 62.66 mol %.

A novel Gram-negative, aerobic, rod-shaped, non-spore-forming bacterial strain, RYU5T, was isolated from a stool sample of an inpatient at a hospital in Okinawa, Japan. The optimal growth temperature of RYU5T was 30 °C. Phylogenetic analysis based on the sequences of housekeeping genes, including the 16S rRNA, rpoB, rpoD and gyrB genes, showed that RYU5T was a member of the Pseudomonas putida group and was located close to Pseudomonas monteilii and P. putida . Whole-genome comparisons, using average nucleotide identity and digital DNA–DNA hybridization, confirmed that strain RYU5T should be classified as a novel species of Pseudomonas . Phenotypic characterization tests showed that utilization of d-mannose, d-serine, l-arabinose and d-fructose could distinguish this strain from other related species of the genus Pseudomonas . Based on genetic and phenotypic evidence, strain RYU5T should be classified as a novel species, for which the name Pseudomonas asiatica sp. nov. is proposed. The type strain is RYU5T (=DSM 107182T, =JCM 32716T), with a DNA G+C content of 62.25 mol%.

Methods for the accomplishment of small-molecule imaging by mass spectrometry are challenged by the need for sample pretreatment steps, such as cryo-sectioning, dehydration, chemical fixation, or application of a matrix or solvent, that must be performed to obtain interpretable spatial distribution data. Furthermore, these steps along with requirements of the mass analyzer such as high vacuum, can severely limit the range of sample types that can be analyzed by this powerful method. Here, we report the development of a laser ablation-direct analysis in real time imaging mass spectrometry approach which couples a 213 nm Nd:YAG solid state UV laser to a direct analysis in a real time ion source and high-resolution time-of-flight mass spectrometer. This platform enables facile determination of the spatial distribution of small-molecules spanning a range of polarities in a diversity of sample types and requires no matrix, vacuum, solvent, or complicated sample pretreatment steps. It furnishes high-resolution data, can be performed under ambient conditions on samples in their native form, and results in little to no fragmentation of analytes. We demonstrate its application through determination of the spatial distribution of molecules involved in the biosynthetic cascade leading to formation of the clinically relevant alkaloids atropine and scopolamine in Datura leichhardtii seed tissue.

Fusions of fatty acids and peptides expand the structural diversity of natural products; however, polyketide/ribosomally synthesized and post-translationally modified peptides (PK/RiPPs) hybrid lipopeptides are relatively rare. Here we report a family of PK/RiPPs called goadvionins, which inhibit the growth of Gram-positive bacteria, and an acyltransferase, GdvG, which catalyses the condensation of the PK and RiPP moieties. Goadvionin comprises a trimethylammonio 32-carbon acyl chain and an eight-residue RiPP with an avionin structure. The positions of six hydroxyl groups and one double bond in the very-long acyl chain were determined by radical-induced dissociation tandem mass spectrometry, which collides radical ion species to generate C-C bond cleavage fragments. GdvG belongs to the Gcn5-related N-acetyltransferase superfamily. Unlike conventional acyltransferases, GdvG transfers a very long acyl chain that is tethered to an acyl carrier protein to the N-terminal amino group of the RiPP moiety. gdvG homologues flanked by PK/fatty acid and RiPP biosynthesis genes are widely distributed in microbial species, suggesting that acyltransferase-catalysed condensation of PKs and RiPPs is a general strategy in biosynthesis of similar lipopeptides.

16S rRNA gene sequences of two type strains belonging to different genera within the suborder Corynebacterineae, namely Hoyosella altamirensis and Amycolicicoccus subflavus, show a similarity of 99.8 %. Therefore, in order to clarify their taxonomic relationship, a polyphasic recharacterization under the same conditions was carried out. The peptidoglycan of H. altamirensis NBRC 109631T and A. subflavus NBRC 109087T was of A1γ type with meso-diaminopimelic acid as their diagnostic diamino acid. Both strains contained MK-8 as the only detected menaquinone, C16 : 0 and C18 : 1ω9c as the major fatty acids, and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as the principal polar lipids. The coincidences of these chemotaxonomic features suggested that H. altamirensis and A. subflavus should be assigned to the same genus. Meanwhile, the average nucleotide identity value between both strains and the results of physiological and biochemical tests indicated that H. altamirensis and A. subflavus should be affiliated to different species. Therefore, according to Rules 38 and 41a of the Bacteriological Code, it is proposed that Amycolicicoccus subflavus Wang et al. 2010 be reclassified as Hoyosella subflava comb. nov. (type strain DQS3-9A1T=CGMCC 4.3532T=DSM 45089T=JCM 17490T=NBRC 109087T) and the descriptions of the genus HoyosellaJurado et al. 2009 and Hoyosella altamirensisJurado et al. 2009 are emended accordingly.

Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% ( n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes.

Two rapidly growing mycobacteria with identical 16S rRNA gene sequences were the subject of a polyphasic taxonomic study. The strains formed a well-supported subclade in the mycobacterial 16S rRNA gene tree and were most closely associated with the type strain of Mycobacterium novocastrense . Single and multilocus sequence analyses based on hsp65, rpoB and 16S rRNA gene sequences showed that strains SN 1900T and SN 1904T are phylogenetically distinct but share several chemotaxonomic and phenotypic features that are are consistent with their classification in the genus Mycobacterium . The two strains were distinguished by their different fatty acid and mycolic acid profiles, and by a combination of phenotypic features. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values for strains SN 1900T and SN 1904T were 61.0 % and 94.7 %, respectively; in turn, the corresponding dDDH and ANI values with M. novocastrense DSM 44203T were 41.4 % and 42.8 % and 89.3 % and 89.5 %, respectively. These results show that strains SN1900T and SN 1904T form new centres of taxonomic variation within the genus Mycobacterium . Consequently, strains SN 1900T (40T=CECT 8763T=DSM 43219T) and SN 1904T (2409T=CECT 8766T=DSM 43532T) are considered to represent novel species, for which the names Mycobacterium lehmannii sp. nov. and Mycobacterium neumannii sp. nov. are proposed. A strain designated as ‘ Mycobacterium acapulsensis’ was shown to be a bona fide member of the putative novel species, M. lehmannii.

A polyphasic study was undertaken to establish the taxonomic position of a non-chromogenic, rapidly growing Mycobacterium strain that had been isolated from sputum. The strain, CECT 8775T, has chemotaxonomic and cultural properties consistent with its classification in the genus Mycobacterium and was distinguished from the type strains of closely related mycobacterial species, notably from Mycobacterium paraense DSM 46749T, its nearest phylogenetic neighbour, based on 16S rRNA, hsp65 and rpoB gene sequence data. These organisms were also distinguished by a broad range of chemotaxonomic and phenotypic features and by a digital DNA–DNA relatedness value of 22.8 %. Consequently, the strain is considered to represent a novel species of Mycobacterium for which the name Mycobacterium eburneum sp. nov is proposed; the type strain is X82T (CECT 8775T=DSM 44358T).

Optical microscopy is generally the first choice to observe microbes and cells. However, its resolution is not always sufficient to reveal specific target structures, such as flagella and pili, which are only nanometers wide. ASEM is an attractive higher resolution alternative, as the sample is observed in aqueous solution at atmospheric pressure. Sample pretreatment for ASEM only comprises simple tasks including fixation, gold labeling, and reagent exchange, taking less than 1 h in total. The lengthy sample pretreatments often required for more classical electron microscopies, such as embedding and dehydration, are unnecessary, and native morphology is preserved. In this study, positively charged nanogold particles were used to label the surfaces of bacteria and cultured animal cells, exploiting their net negative surface charge. After gold enhancement to increase the size of the nanogold particles, ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella. This natural shape contrasts starkly with images of dried flagella recorded by standard SEM. Positively charged nanogold labeled the plasma membrane of cultured COS7 cells, and after enhancement allowed filopodia as thin as 100 nm in diameter to be clearly visualized. Based on these studies, ASEM combined with positively charged nanogold labeling promises to become an important tool for the study of cell morphology and dynamics in the near future. Microsc. Res. Tech. 77:153–160, 2014. © 2013 Wiley Periodicals, Inc.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for the analysis of not only biological materials but also synthetic polymers. A mass spectrum obtained by MALDI-MS was found to have peaks at equal intervals on the m/z axis. The chemical structure of many types of biological compounds contain repeating units that have exactly the same elemental composition. If these repeated peaks could be identified based on the m/z interval value, such analyses could be optimized even further from the point of view of chemical structure. In this study, we describe a system for visualizing such data that has the capability of analyzing the repeated peaks on the m/z axis. The system, mzRepeat, was developed and evaluated by applying the mass spectrum of polyethylene glycol and mycolic acid, which was obtained using a JMS-S3000 Spiral-TOFMS system. Because mzRepeat can systematically show a mass spectrum that includes different numbers of repeating units, it has the potential for use in analyzing these types of molecules.

Polyphenolic compounds from chestnut astringent skin (CAS) were purified by dialysis, using Diaion HP-20 and Sephadex LH-20 columns. During purification, specific α-amylase inhibitory activities were increased about 3.4-fold, and the 50% inhibition value was 5.71 μg/mL in the Sephadex LH-20 fraction (SE-fraction). The SE-fraction contained about 67% of the total polyphenols, 57.3% of the flavanol-type tannins, and 51.3% of the procyanidins. Strong antioxidant activity was observed in the SE-fraction. Oral administration of the SE-fraction in rats fed corn starch significantly suppressed an increase in blood glucose levels. The SE-fraction contained gallic acid and ellagic acid. The MALDI-TOF spectrum showed a peak series exhibiting a mass increment of 288 Da, reflecting the variation in the number of catechin/epicatechin units. Our results suggest CAS contains polyphenols with strong α-amylase inhibitory activity. The data also suggest CAS polyphenols might be oligomeric proanthocyanidins with gallic acid and ellagic acid.

Rhodococcus erythropolis strains characterized as antibiotic producers can be classified into three groups according to their antibiotic spectrum and growth compatibility. Due to their high genotypic similarity, the taxonomic relationship of these strains has not been elucidated. In this study, ribosomal protein profiling using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was employed to classify twenty-one strains of R. erythropolis (15 antibiotic producers and 6 non-antibiotic producers). In the first step in this method, a total of 30 intense peaks observed for purified ribosomal subunit proteins of the type strain (R. erythropolis JCM 3201(T)) were selected as the reference peaks. The mass spectra observed for the cell lysates of each sample strain were then checked as to whether peaks were observed at the same masses of the reference peaks. The results of peak matching were processed by cluster analysis, generating a dendrogram. Four major clusters of the R. erythropolis strains corresponded to three antibiotic groups and the non-antibiotic group. Furthermore, the topology of the dendrogram was highly comparable with the phylogenetic tree based on DNA gyrase subunit B gene (gyrB) sequencing. These results indicate that our proposed ribosomal protein profiling method using MALDI-MS is a potentially reliable and sufficiently high-throughput technique for the taxonomic analysis of closely related bacterial strains without using DNA sequence information.

Rapid identification and classification of bacteria by mass spectrometry (MS) have been expected to play important roles in various fields such as food industries, biomedical assays, and environmental analysis. In this review, the historical progress of various mass spectrometric approaches from pyrolysis-MS to matrix-assisted laser desorption/ionization (MALDI)-MS and their features are outlined. Then, the details of the development of bacterial analysis based on MALDI-MS are discussed. Among these, recent progress in MALDI-MS-based identification and classification of bacteria using ribosomal proteins as biomarkers is described in brief.

Fifteen soil samples from the edge of a creek were investigated for alkylphenol polyethoxylate (APEO) degrading ability. Ten soil samples possessed PEG chain shortening activity and the other soil samples oxidized the terminal hydroxyl group to the corresponding carboxyl group. Colonies of APEO-degrading bacteria having slight morphological differences were isolated and the bacteria were grouped by MALDI-MS patterns, PCR-RFLP patterns, and sequence analysis of the gyrB gene. The isolates were divided into three groups by typing with the MALDI-MS pattern. PCR-RFLP analysis using 41f and 1066r primer sets and three kinds of restriction enzyme (Alul, HaeIII, HhaI) indicated that the isolates belonged to Pseudomonas sp., but all isolates gave the same RFLP pattern. Sequence analysis of gyrB gene divided the isolates into two groups. The grouping results by MALDI-MS spectra and gyrB sequence were identical and represent the APEO degrading ability of the isolates.

Ribosomal subunit proteins of eight genome-sequenced lactic acid bacteria (LAB) were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to provide a guideline for selecting reliable biomarkers used for the rapid identification of LAB. Subunit proteins with molecular weight over ca. 20,000 were typically difficult to detect. This phenomenon appeared to be due to the mass discrimination effect in MALDI-MS. Conserved post-translational modifications excluding N-terminal methionine loss were investigated by comparing the mass differences from sequence masses calculated from the amino acid sequences of the proteins with the post-translational modifications. Various errors in the amino acid sequences of the subunit proteins registered in protein databases could be detected by comparing amino acid sequences, and corrected by inspecting gene sequences. By eliminating the subunit proteins with higher molecular weight and post-translational modifications as well as other difficult to detect subunit proteins, 27 subunit proteins were finally selected as the reliable biomarkers.

A new method for phylogenetic classification of bacterial strains using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is proposed. This method was developed using a bioinformatics-based approach to the rapid identification of bacteria as previously proposed by Demirev and co-workers, which uses ribosomal proteins composed of approximately 50 subunit proteins as biomarkers. Although the amino acid sequences of ribosomal proteins are highly conserved, slight sequence variations can occur at the strain level. Since ribosomal subunit proteins are a complex of housekeeping proteins that have different phylogenetic evolution rates, sequence variation detected as mass differences by MALDI-MS may be useful for the phylogenetic classification of bacteria at strain level. In our proposed method, the first step is the selection of reliable biomarkers through characterization of the expressed ribosomal subunit proteins of a reference strain (usually a genome-sequenced strain) by MALDI-MS. The observed masses in the MALDI mass spectra of cell lysates of sample strains are then compared with the biomarker masses of the reference strain. The biomarkers for each sample strain were designated as present or absent at the reference masses, indicated by 1 or 0, respectively, which were summarized in a table. This table is processed by cluster analysis, generating a phylogenetic tree. In this study, the success of this approach was confirmed by classification of Pseudomonas putida strains because its classification is much more complicated than that of other bacterial strains. Forty-three reliable biomarkers were selected from ribosomal sub-unit proteins of a genome-sequenced strain, P. putida KT2440. The numbers and kinds of biomarkers observed for 16 strains of P. putida, including different biovars, were markedly different, reflecting the variety of the strains. The classification results by the proposed method were highly comparable to those based on the DNA gyrase subunit B gene (gyrB) sequence analysis, suggesting our proposed method would be a useful high-throughput method for phylogenetic classification of newly isolated bacteria.

Rapid identification of bacteria by a bioinformatics-based approach, which processes the mass spectra observed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), relies on the calculated masses of ribosomal subunit proteins as biomarkers predicted from amino acid sequences found in protein sequence databases. To verify the actual state of the registered sequence information, a simple intact protein analysis by MALDI-MS using cell lysates as samples was applied to the characterization of ribosomal proteins from genome-sequenced Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains. This method avoided the risk of loss of some subunit proteins and the formation of disulfide bonds during the purification of ribosomal proteins. By comparing this with the MALDI mass spectra of different strains and carrying out manual inspection of sequence information, a total of five errors in N-terminal amino acid sequences were identified. After sequence correction, approximately 40 out of 53 subunit proteins could be assigned, considering N-terminal methionine loss only as a post-translational modification. These show promise for use as practical biomarkers for the rapid identification of S. thermophilus and L. bulgaricus. After verification of these amino acid sequences, mass differences relative to those of genome-sequenced strains have the potential for distinguishing bacteria at the strain level.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is beginning to play an important role in rapid identification and classification of industrially useful or pathogenic bacteria. Instead of conventional mass spectral fingerprint approaches, MALDI-MS identification of bacteria using ca. 50 ribosomal subunit proteins as biomarkers has been proposed. In this bioinformatics-based method, the masses of ribosomal subunit proteins in bacterial lysates observed by MALDI-MS are compared with the calculated masses predicted from the amino acid sequences registered on protein databases such as Swiss-Prot. Therefore, taxonomic identification of bacteria at species level would be ideally performed. Considering the possible mismatch between the actual and calculated masses of the biomarkers, we further developed a highly reliable method applicable to bacterial classification at strain level by detailed characterization of expressed ribosomal subunit proteins of several bacteria. With our developed method using a verified mass list of expressed ribosomal proteins of genome-sequenced strains as a reference, bacterial samples isolated from soil could be successfully classified at strain level. As an application, detection of cross-contamination of culture collections was also described.

マトリックス支援レーザー脱離イオン化質量分析法（MALDI-MS）により観測されるリボソームタンパク質をバイオマーカーとした迅速分析法を用いて，Lactobacillus sakeiであると考えられている市販の低温発酵乳酸菌スターターD-1001株，及び生化学的な特性が類似している低温下でも増殖可能な乳酸菌15菌株の同定・分類を試みた．本研究に用いた16菌株の乳酸菌は，MALDIマススペクトルで観測されたリボソームタンパク質の観測質量から，いずれもL. sakeiであることが示され，16S rRNA遺伝子の部分塩基配列解析の結果を支持した．更にバイオマーカーとして選択したピークを用いて，単離菌株の株レベルでの分類を試みた．バイオマーカーピークと同じm/z値に観測されているピークの有無を調べてクラスター解析を行った結果，16種類の乳酸菌を4つのグループに分類することができた．

Co-culture of Streptomyces with mycolic acid-containing bacteria (MACB), which we termed "combined-culture," alters the secondary metabolism pattern in Streptomyces and has been a useful method for the discovery of bioactive natural products. In the course of our investigation to identify the inducing factor(s) of MACB, we previously observed that production of pigments in Streptomyces lividans was not induced by factors such as culture extracts or mycolic acids. Although dynamic changes occurred in culture conditions because of MACB, the activation of pigment production by S. lividans was observed in a limited area where both colonies were in direct contact. This suggested that direct attachment of cells is a requirement and that components on the MACB cell membrane may play an important role in the response by S. lividans. Here we examined whether this response was influenced by dead MACB that possess intact mycolic acids assembled on the outer cell membrane. Formaldehyde fixation and γ-irradiation were used to prepare dead cells that retain their shape and mycolic acids of three MACB species: Tsukamurella pulmonis, Rhodococcus erythropolis, and Rhodococcus opacus. Culturing tests verified that S. lividans does not respond to the intact dead cells of three MACB. Observation of combined-culture by scanning electron microscopy (SEM) indicated that adhesion of live MACB to S. lividans mycelia were a significant interaction that resulted in formation of co-aggregation. In contrast, in the SEM analysis, dead cells were not observed to adhere. Therefore, direct attachment by live MACB cells is proposed as one of the possible factors that causes Streptomyces to alter its specialized metabolism in combined-culture.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is beginning to play an important role in a rapid discrimination of bacteria, where ribosomal proteins composed of more than 50 subunits is used as biomarkers. In this study, this technique was applied to the maintenance of bacterial culture collections. It was confirmed that most of the biomarker peaks could be observed in the whole cell lysate of model lactic acid bacteria, which was prepared simply by bead-beating and centrifugation. The mass differences of specific ribosomal subunit proteins observed on the MALDI mass spectra allowed us to discriminate two lactic acid bacteria at a subspecies level difference (Lactococcus lactis subsp. lactis and Lc. lactis subsp. cremoris). By comparing the MALDI mass spectrum of the stored culture of Lc. lactis subsp. lactis with that of the pure culture as a reference, it could be easily found that the stored culture was cross-contaminated with other lactic acid bacteria.

Mycolic acids (MAs) are characteristic components of bacteria in the suborder Corynebacterineae, such as Mycobacterium. MAs are categorized into subclasses based on their functional bases (cyclopropane ring, methoxy, keto, and epoxy group). Since MAs have heterogeneity among bacterial species, analyzing of MAs are required in the chemotaxonomic field. However, their structural analysis is not easy because of their long carbon-chain lengths and several functional groups. In this study, total fatty acid (FA) methyl ester (ME) fraction of M. tuberculosis H37Rv was analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) with a spiral ion trajectory (MALDI spiral-TOFMS). The distributions of carbon-chain length and their relative peak intensities were confirmed with those obtained by analysis of each subclass fraction which was separated from total FA ME fraction using thin-layer chromatography (TLC). The observed major peaks were reliably assigned as MAs owing to the high mass accuracy (error

High-resolution mass spectrometry (HRMS) continues to play an important role in the compositional characterization of larger organic molecules. In the field of polymer characterization, however, the application of HRMS has made only slow progress because of lower compatibility between matrix-assisted laser desorption/ionization (MALDI) and ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS). In this study, a newly developed type of MALDI high-resolution time-of-flight mass spectrometry (TOFMS) with a spiral ion trajectory (MALDI spiral-TOFMS) was applied to the structural and compositional characterization of polymers. To create a graphical distribution of polymer components on a two-dimensional plot converted from complex mass spectra, we adopted a slightly modified Kendrick mass defect (KMD) analysis based on accurate masses determined using spiral-TOFMS. By setting the Kendrick mass scale based on the mass of the repeating units of a given polymer, components with common repeat units lined up in the horizontal direction on the KMD plot, whereas those components with different structures were shifted vertically. This combination of MALDI spiral-TOFMS measurement and KMD analysis enabled the successful discrimination of the polymer components in a blend of poly(alkylene oxide)s, the compositional analysis of poly(ethylene oxide)/poly(propylene oxide) block copolymers, and profiling of the end-group distribution of poly(ε-caprolactone)s synthesized under different conditions. ᅟ

Optical microscopy is generally the first choice to observe microbes and cells. However, its resolution is not always sufficient to reveal specific target structures, such as flagella and pili, which are only nanometers wide. ASEM is an attractive higher resolution alternative, as the sample is observed in aqueous solution at atmospheric pressure. Sample pretreatment for ASEM only comprises simple tasks including fixation, gold labeling, and reagent exchange, taking less than 1 h in total. The lengthy sample pretreatments often required for more classical electron microscopies, such as embedding and dehydration, are unnecessary, and native morphology is preserved. In this study, positively charged nanogold particles were used to label the surfaces of bacteria and cultured animal cells, exploiting their net negative surface charge. After gold enhancement to increase the size of the nanogold particles, ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella. This natural shape contrasts starkly with images of dried flagella recorded by standard SEM. Positively charged nanogold labeled the plasma membrane of cultured COS7 cells, and after enhancement allowed filopodia as thin as 100 nm in diameter to be clearly visualized. Based on these studies, ASEM combined with positively charged nanogold labeling promises to become an important tool for the study of cell morphology and dynamics in the near future.

Mycolic acids have been used as important chemotaxonomic markers. In this study, a newly developed matrix-assisted laser desorption/ionization time-of-flight mass spectrometer with a spiral ion trajectory (MALDI spiral-TOFMS) was applied to the characterization of mycolic acids of three type strains of validated species belonging to the genus Dietzia (D. papillomatosis 105045(T), D. kunjamensis NBRC 105042(T) and D. timorensis NBRC 104184(T)), by analysis of total fatty acid methyl ester fractions. In addition, owing to the high mass-resolving power of MALDI spiral-TOFMS, adjacent peaks (0.036 Da mass differences) were successfully separated, and weak peaks corresponding to oxygenated mycolic acids were detected. For all samples, the distributions of carbon-chain lengths were mainly in the range of C30-C42 and the average number of carbon-chain lengths was about 37, which agreed reasonably well with reported results for the genus Dietzia. The number of double bonds and/or cyclopropane rings was 0-2. Relative peak intensities of each mycolic acid methyl ester were used to compare the mycolic acids of the three strains. The mycolic acids of D. papillomatosis and D. kunjamensis were characterized by a high content of mycolic acids with 0-1 double bond or cyclopropane ring and an almost equal content of mycolic acids with odd- and even-numbered carbon-chain lengths. In contrast, mycolic acids of D. timorensis were characterized by a high content of mycolic acids with 1-2 double bonds and/or cyclopropane rings with an even-numbered carbon-chain length. By using MALDI spiral-TOFMS, mycolic acids from three type strains of the genus Dietzia were characterized easily and rapidly.

Mass spectrometry is widely used for identifying molecules that have similar structures. A mass spectrum obtain from lipids using MALDI-MS (matrix-assisted laser desorption/ionization-mass spectrometer) was observed to have peaks at equal intervals on the m/z axis. If these repeated peaks can be collectively shown, analysis can be optimized even further. In this study, we propose a visualization system for analyzing repeated observed peaks on the m/z axis. The system, mzRepeat, was developed and evaluated by analyzing butter. A feature of mzRepeat is that ions that differ only in number of repeating structures are shown collectively. It is especially effective for analysis of many peaks from the samples that contain several molecular species.

We have proposed a rapid phylogenetic classification at the strain level by MALDI-TOF MS using ribosomal protein matching profiling. In this study, the S10-spc-alpha operon, encoding half of the ribosomal subunit proteins and highly conserved in eubacterial genomes, was selected for construction of the ribosomal protein database as biomarkers for bacterial identification by MALDI-TOF MS analysis to establish a more reliable phylogenetic classification. Our method revealed that the 14 reliable and reproducible ribosomal subunit proteins with less than m/z 15,000, except for L14, coded in the S10-spc-alpha operon were significantly useful biomarkers for bacterial classification at species and strain levels by MALDI-TOF MS analysis of genus Pseudomonas strains. The obtained phylogenetic tree was consisted with that based on genetic sequence (gyrB). Since S10-spc-alpha operons of genus Pseudomonas strains were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains, the ribosomal subunit proteins encoded in S10-spc-alpha operon were suitable biomarkers for construction and correction of the database. MALDI-TOF MS analysis using these 14 selected ribosomal proteins is a rapid, efficient, and versatile bacterial identification method with the validation procedure for the obtained results.

Mycolic acids (MAs) are characteristic components of bacteria in the suborder Corynebacterineae, such as Mycobacterium. MAs are categorized into subclasses based on their functional bases (cyclopropane ring, methoxy, keto, and epoxy group). Since MAs have heterogeneity among bacterial species, analyzing of MAs are required in the chemotaxonomic field. However, their structural analysis is not easy because of their long carbon-chain lengths and several functional groups. In this study, total fatty acid (FA) methyl ester (ME) fraction of M. tuberculosis H37Rv was analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) with a spiral ion trajectory (MALDI spiral-TOFMS). The distributions of carbon-chain length and their relative peak intensities were confirmed with those obtained by analysis of each subclass fraction which was separated from total FA ME fraction using thin-layer chromatography (TLC). The observed major peaks were reliably assigned as MAs owing to the high mass accuracy (error

For rapid identification of bacteria by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), a bioinformatics approach using ribosomal subunit proteins as biomarkers has been proposed. This method compares the observed masses for biomarkers with calculated masses as predicted from the amino acid sequences registered on protein databases. To evaluate this approach, the expressed ribosomal proteins of a genome-sequenced bacterium, Lactobacillus plantarum NCIMB 8826, were characterized as a model sample. The protein expression of 42 ribosomal subunit proteins, together with 10 ribosome-associated proteins in the isolated ribosome fraction, was confirmed through two-dimensional gel electrophoresis combined with peptide mass fingerprinting. The observed masses of the proteins in the isolated ribosome fraction were then determined by MALDI-MS. We preliminarily selected 44 biomarkers whose observed masses were matched with the calculated masses predicted from the amino acid sequence registered in the protein databases by considering N-terminal methionine loss only. Of these, the finally selected reliable biomarkers were 34 proteins including 31 ribosomal subunit proteins and 3 ribosome-associated proteins that could be observed in the MALDI mass spectra of the cell lysate sample. These biomarkers were usable in MALDI-MS characterization of two industrial L. plantarum cultures.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

The structural characterization of copolymers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) remains a challenging task, since their random comonomer distribution creates very complicated mass spectra. In this study, a high-resolution TOF mass spectrometer with a spiral ion trajectory was applied to the structural and compositional characterization of free radical copolymerized poly(methyl methacrylate-co-tert-butyl methacrylate), poly(MMA-co-tBMA)s in ethyl lactate acting as a chain transfer agent. Virtually complete peak assignments of the isobaric components within the poly(MMA-co-tBMA)s served to identify the end-group combinations and copolymer compositions of individual copolymer components, allowing the distributions of comonomer compositions and six types of end-group combinations to be evaluated.