MS Identification Research Articles

Deficiency of polypeptide N-acetylgalactosamine transferase 9 contributes to a risk for Parkinson's disease via mitochondrial dysfunctions

Polypeptide N-acetylgalactosamine transferase 9 (GALNT9) catalyzes the initial step of mucin-type O-glycosylation via linking N-acetylgalactosamine (GalNAc) to serine/threonine in a protein. To unravel the association of GALNT9 with Parkinson's disease (PD), a progressive neurodegenerative disorder, GALNT9 levels were evaluated in the patients with PD and mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and statistically analyzed based on the GEO datasets of GSE114918 and GSE216281. Glycoproteins with exposing GalNAc were purified using lectin affinity chromatography and identified by LC-MS/MS. The influence of GALNT9 on cells was evaluated via introducing a GALNT9-specific siRNA into SH-SY5Y cells. Consequently, GALNT9 deficiency was found to occur under PD conditions. GALNT9 silencing contributed to a causative factor in PD pathogenesis via reducing the levels of intracellular dopamine, tyrosine hydroxylase and soluble α-synuclein, and promoting α-synuclein aggregates. MS identification revealed 14 glycoproteins. 5 glycoproteins, including ACO2, ATP5B, CKB, CKMT1A, ALDOC, were associated with energy metabolism. GALNT9 silencing resulted in mitochondrial dysfunctions via increasing ROS accumulation, mitochondrial membrane depolarization, mPTPs opening, Ca2+ releasing and activation of the CytC-related apoptotic pathway. The dysfunctional mitochondria then triggered mitophagy, possibly intermediated by adenine nucleotide translocase 1. Our study suggests that GALNT9 is potentially developed into an auxiliary diagnostic index and therapeutic target of PD.

Comparison of MALDI-TOF mass spectrometry and 16S rDNA sequencing for identification of environmental bacteria: a case study of cave mussel-associated culturable microorganisms.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is routinely used as a rapid and cost-effective method for pathogen identification in clinical settings. In comparison, its performance in other microbiological fields, such as environmental microbiology, is still being tested, although isolates of environmental microbes are essential for in-depth in vivo studies of their biology, including biotechnological applications. We investigated the applicability of MALDI-TOF MS for the identification of bacterial isolates from a highly oligotrophic environment-Dinaric Karst caves, which likely harbor specific microorganisms. We cultured bacteria from the shell surface of the endemic mussel Congeria jalzici, one of the three known cave mussels in the world that lives in the Dinaric karst underground. The bacterial isolates were obtained by swabbing the shell surface of mussels living in microhabitats with different amounts of water: 10 air-exposed mussels, 10 submerged mussels, and 10 mussels in the hygropetric zone. A collection of 87 pure culture isolates was obtained, mostly belonging to the phylum Bacillota (72%), followed by Pseudomonadota (16%), Actinomycetota (11%), and Bacteroidota (1%). We compared the results of MALDI-TOF MS identification (Bruker databases DB-5989 and version 11, v11) with the results of 16S rDNA-based phylogenetic analysis, a standard procedure for bacterial identification. Identification to the genus level based on 16S rDNA was possible for all isolates and clearly outperformed the results from MALDI-TOF MS, although the updated MALDI-TOF MS database v11 gave better results than the DB-5989 version (85% versus 62%). However, identification to the species-level by 16S rDNA sequencing was achieved for only 17% of isolates, compared with 14% and 40% for the MALDI-TOF MS databases DB-5989 and v11 database, respectively. In conclusion, our results suggest that continued enrichment of MALDI-TOF MS libraries will result with this method soon becoming a rapid, accurate, and efficient tool for assessing the diversity of culturable bacteria from different environmental niches.

Ceiba pentandra ethyl acetate extract improves doxorubicin antitumor outcomes against chemically induced liver cancer in rat model: a study supported by UHPLC-Q-TOF-MS/MS identification of the bioactive phytomolecules.

Hepatocellular carcinoma (HCC) is a prevalent cancer worldwide. Late-stage detection, ineffective treatments, and tumor recurrence contribute to the low survival rate of the HCC. Conventional chemotherapeutic drugs, like doxorubicin (DOX), are associated with severe side effects, limited effectiveness, and tumor resistance. To improve therapeutic outcomes and minimize these drawbacks, combination therapy with natural drugs is being researched. Herein, we assessed the antitumor efficacy of Ceiba pentandra ethyl acetate extract alone and in combination with DOX against diethylnitrosamine (DENA)-induced HCC in rats. Our in vivo study significantly revealed improvement in the liver-function biochemical markers (ALT, AST, GGT, and ALP), the tumor marker (AFP-L3), and the histopathological features of the treated groups. A UHPLC-Q-TOF-MS/MS analysis of the Ceiba pentandra ethyl acetate extract enabled the identification of fifty phytomolecules. Among these are the dietary flavonoids known to have anticancer, anti-inflammatory, and antioxidant qualities: protocatechuic acid, procyanidin B2, epicatechin, rutin, quercitrin, quercetin, kaempferol, naringenin, and apigenin. Our findings highlight C. pentandra as an affordable source of phytochemicals with possible chemosensitizing effects, which could be an intriguing candidate for the development of liver cancer therapy, particularly in combination with chemotherapeutic drugs.

Diversity and dose-dependent allelopathic potential of volatile sesquiterpenes from root extracts of Chrysanthemum morifolium cultivars

Chrysanthemum × morifolium Ramat. (chrysanthemum) is rich in terpenoids in both aboveground and belowground parts with multiple biological functions. This study aims to comprehensively assess the diversity of root-derived terpenoids and their potential role in plant-plant interaction in chrysanthemums. By GC–MS identification, we revealed the intraspecific variation of terpenoid components dominated by sesquiterpenes in the root extracts among 26 chrysanthemum cultivars with different application types (including cut-flower, ground-cover and tea cultivars). The total terpenoid content in tea cultivars was generally higher than that in ground-cover and cut-flower cultivars. In this context, the correlation between the terpenoids from root extracts and allelopathic effect was established. The root extract of tea, cut-flower and ground-cover cultivars could universally promote the root length of Lactuca sativa seedling without significant effect on bud length. Moreover, the root extracts of tea and cut-flower cultivars showed no significant effects on the bud length and root length of Echinochloa crus-galli seedlings, whereas the root extract of ground-cover cultivars could significantly promote the bud length and inhibit the root length of E. crus-galli seedlings. OPLS analysis successfully correlated the content of sesquiterpenes (β-farnesene, β-elemene, β-bisabolene, neollocimene, β-sesquiphellandrene and α-longipinene) with bud and root length of L. sativa and E. crus-galli seedlings. Additionally, root extracts of 3 representative cultivars in each application type displayed dose-dependent effect in the seed germination of L. sativa. These findings provide a scientific basis for the development of eco-friendly herbicides\\plant growth regulator and the arrangement of reasonable farming systems in chrysanthemum.

Preparation, Identification, and Digestive Stability of Antioxidant Peptides From Chlorella vulgaris

Chlorella, a single‐celled green algae rich in proteins, holds promise as a source of bioactive peptides. This research aimed to isolate an antioxidant peptide from Chlorella, examining its stability through digestion. Using Response Surface Methodology (RSM), we identified optimal hydrolysis conditions for Chlorella Protein Hydrolysate (CPH): 55°C, pH 8.75, and an enzyme‐to‐substrate ratio of 2.15%. These conditions led to CPH with notable radical scavenging activity against 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) (53.19 ± 0.99%), 2,2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) (76.86 ± 0.28%), hydroxyl radical (53.39 ± 1.74%), and Fe2+ chelating rates of 22.56 ± 2.97%. Subsequently, peptides under 3 kDa isolated by ultrafication proved particularly stable and antioxidative after in vitro digestion. In addition, further purify to obtain component C1 with DPPH radical scavenging ability of 59.62 ± 1.29% and an Fe2+ chelating ability of 49.78 ± 1.55%. Subsequent LC‐MS/MS identification and virtual screening led to the discovery of a stable and antioxidative peptide, Ser‐Gly‐His‐His‐Lys‐Pro‐Leu (SGHHKPL). The study provides the theoretical basis for the development and utilization of chlorella.

Wastewater and environmental sampling holds potential for antimicrobial resistance surveillance in food-producing animals - a pilot study in South African abattoirs.

Antimicrobial resistance (AMR) poses a significant global One Health challenge that causes increased mortality and a high financial burden. Animal production contributes to AMR, as more than half of antimicrobials are used in food-producing animals globally. There is a growing body of literature on AMR in food-producing animals in African countries, but the surveillance practices across countries vary considerably. This pilot study aims to explore the potential of wastewater and environmental surveillance (WES) of AMR and its extension to the veterinary field. Floor drainage swab (n = 18, 3/abattoir) and wastewater (n = 16, 2-3/abattoir) samples were collected from six South African abattoirs that handle various animal species, including cattle, sheep, pig, and poultry. The samples were tested for Extended-Spectrum Beta-Lactamase (ESBL) and Carbapenemase-producing Enterobacterales, Methicillin-Resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococci (VRE), and Candida auris by using selective culturing and MALDI-TOF MS identification. The phenotype of all presumptive ESBL-producing Escherichia coli (n = 60) and Klebsiella pneumoniae (n = 24) isolates was confirmed with a disk diffusion test, and a subset (15 and 6 isolates, respectively), were further characterized by whole-genome sequencing. In total, 314 isolates (0-12 isolates/sample) withstood MALDI-TOF MS, from which 37 species were identified, E. coli and K. pneumoniae among the most abundant. Most E. coli (n = 48/60; 80%) and all K. pneumoniae isolates were recovered from the floor drainage samples, while 21 presumptive carbapenem-resistant Acinetobacter spp. isolates were isolated equally from floor drainage and wastewater samples. MRSA, VRE, or C. auris were not found. All characterized E. coli and K. pneumoniae isolates represented ESBL-phenotype. Genomic analyses revealed multiple sequence types (ST) of E. coli (n = 10) and K. pneumoniae (n = 5), including STs associated with food-producing animals globally, such as E. coli ST48 and ST10 and K. pneumoniae ST101. Common beta-lactamases linked to food-producing animals, such as bla CTX-M-55 and bla CTX-M-15, were detected. The presence of food-production-animal-associated ESBL-gene-carrying E. coli and K. pneumoniae in an abattoir environment and wastewater indicates the potential of WES in the surveillance of AMR in food-producing animals. Furthermore, the results of this pilot study encourage studying the topic further with refined methodologies.

A Systematic Study of Yiqi Qubai Standard Decoction for Treating Vitiligo Based on UPLC-Q-TOF/MS Combined with Chemometrics, Molecular Docking, and Cellular and Zebrafish Assays.

The Yiqi Qubai (YQ) formula is a hospital preparation for treating vitiligo in China that has had reliable efficacy for decades. The formula consists of four herbs; however, the extraction process to produce the formula is obsolete and the active ingredients and mechanisms remain unknown. Therefore, in this paper, fingerprints were combined with the chemometrics method to screen high-quality herbs for the preparation of the YQ standard decoction (YQD). Then, the YQD preparation procedure was optimized using response surface methodology. A total of 44 chemical constituents, as well as 36 absorption components (in rat plasma) of YQD, were identified via UPLC-Q-TOF/MS. Based on the ingredients, the quality control system of YQD was optimized by establishing the SPE-UPLC-Q-TOF/MS identification method and the HPLC quantification method. Network pharmacological analysis and molecular docking showed that carasinaurone, calycosin-7-O-β-d-glucoside, methylnissolin-3-O-glucoside, genkwanin, akebia saponin D, formononetin, akebia saponin B, and apigenin may be the key active components for treating vitiligo; the core targets associated with them were AKT1, MAPK1, and mTOR, whereas the related pathways were the PI3K-Akt, MAPK, and FoxO signaling pathways. Cellular assays showed that YQD could promote melanogenesis and tyrosinase activity, as well as the transcription and expression of tyrosinase-associated proteins (i.e., TRP-1) in B16F10 cells. In addition, YQD also increased extracellular tyrosinase activity. Further efficacy validation showed that YQD significantly promotes melanin production in zebrafish. These may be the mechanisms by which YQD improves the symptoms of vitiligo. This is the first systematic study of the YQ formula that has optimized the standard decoction preparation method and investigated the active ingredients, quality control, efficacy, and mechanisms of YQD. The results of this study lay the foundations for the clinical application and further development of the YQ formula.

Morphological, molecular, and MALDI-TOF MS identification of bed bugs and associated Wolbachia species from Cameroon

After vanishing from the public eye for more than 50 years, bed bugs have resurged to become one of the most widely discussed and heavily researched insect pests in the world. This study presents the basic information of infestations of tropical bed bugs, Cimex hemipterus (Hemiptera: Cimicidae), in Cameroon. A total of 248 immature stage and adult bed bug specimens were collected from households and a travel agency in Yaoundé and Douala, Cameroon. The ability of MALDI-TOF MS to identify bed bugs was tested using heads for adults and cephalothoraxes for immature stages. Microorganism screening was performed by qPCR and confirmed by regular PCR and sequencing. Based on morphometrical criteria, four stages of immature bed bugs are represented. Of the 248 bed bug specimens morphologically identified as Cimex hemipterus, 246 (77 males, 65 females and 104 immature specimens) were submitted to MALDI-TOF MS analysis. Of the 222 adults and immature specimens tested, 122 (59.9 %) produced good quality MALDI-TOF MS spectra (35 adults and 87 immature specimens). Blind testing allowed species level identification of 98.21 % of adult and immature C. hemipterus. Among the bacteria tested, only Wolbachia DNA was found in 12/246 (4.8 %) bed bugs. More surveys in the country are warranted to assess the true level of bed bug infestations, in order to take appropriate action for their control.

Evaluation of Immulex S. pneumoniae Omni test for the direct detection of S. pneumoniae from positive blood cultures

Rapid and early identification of Streptococcus pneumoniae from positive blood cultures is crucial for the management of patients with bloodstream infections (BSI). Many identification systems in microbiology laboratories have difficulty differentiating S. pneumoniae from other closely related species in the Streptococcus mitis group. To overcome this limitation, we developed a rapid workflow in our laboratory combining direct MALDI-TOF MS identification with the Immulex S. pneumoniae Omni test (SSI Diagnostica, Denmark) for rapid detection of S. pneumoniae directly from positive blood cultures. The workflow was evaluated using 51 Streptococcus isolates. Compared to conventional biochemical testing, our new workflow demonstrates 100 % specificity and sensitivity for the detection and differentiation of S. pneumoniae from other closely related species. Our new workflow is accurate, cost-effective, and can easily be implemented in microbiology laboratories that already perform direct MALDI-TOF identification from positive blood cultures to improve the management of patients with invasive pneumococcal disease. ImportanceInvasive pneumococcal disease remains a major public health problem worldwide. Reducing the time to identify Streptococcus pneumoniae in positive blood cultures allows patients to be treated sooner with more targeted and effective antibiotics. We evaluated a two-step protocol where positive blood cultures are first tested directly by MALDI-TOF MS and any samples containing Streptococcus species are tested by Immulex S. pneumoniae Omni test to both detect and differentiate S. pneumoniae from other closely related Streptococcus species. Our study results showed 100 % sensitivity and specificity, and a much faster turn-around time than conventional methods.

Identification and validation of active ingredient in cerebrotein hydrolysate-I based on pharmacokinetic and pharmacodynamic studies.

Cerebrotein hydrolysate-1 (CH-1), a mixture of small peptides, polypeptides and various amino acids derived from porcine brain, has been widely used in the treatment of cerebral injury. However, the bioactive composition and pharmacokinetics of CH-1 are still unexplored due to their complex constitutes and extremely low concentrations in vivo Herein, NanoLC-Orbitrap-Fusion-Lumos-Tribrid-MS/MS was firstly used to qualitatively analyze the components of CH-1. A total of 1347 peptides were identified, of which 43 peptides were characterized by high MS intensity and identification accuracy. We then innovatively synthesized 4 main peptides for activity verification, and the results suggested that Pep72 (NYEPPTVVPGGDL) had the strongest neuroprotective effect on both in vivo and in vitro models. Next, a quantitative method for Pep72 was established based on LC-MS/MS with the aid of Skyline software, and then used in its pharmacokinetic studies. The results revealed that Pep72 had a high elimination rate and low exposure in rats. In addition, hCMEC/D3 based in vitro model was built and firstly used to investigate the transport of Pep72. We found that Pep72 had extremely low blood-brain barrier (BBB) permeability, and was not a substrate of efflux transporters. The biotransformation of Pep72 in rat fresh plasma and tissues was investigated to explore the contradiction between pharmacokinetics and efficacy. A total of 11 main metabolites were structurally identified, with PGGDL and EPPTVPGGDL being the main metabolites of Pep72. Notably, metalloproteinase and cysteine protease were confirmed to be the main enzymes mediating Pep72 metabolism in rat tissues. Significance Statement NanoLC-Orbitrap-Fusion-Lumos-Tribrid-MS/MS was firstly used to qualitatively analyze the components of CH, and one main peptide Pep72 (NYEPPTVVPGGDL) was innovatively synthesized and firstly found to have the strongest neuroprotective effect among 1347 peptides identified from CH, followed by its pharmacokinetic studies. Our study is the first time to identify and verify the active ingredient of CH from the perspective of pharmacokinetics and pharmacodynamics, and provides a systematic technical platforms and strategies for the active substance research of other protein hydrolysates.

Aureimonas altamirensis: First Isolation from a Chicken Slaughterhouse in Italy Followed by Genotype and Phenotype Evaluations

The presence of foodborne pathogens in meat is linked to several contamination sources, and the slaughterhouse environment represents a relevant reservoir of contamination. Aureimonas altamirensis is a Gram-negative bacteria associated with different isolation sources, including human clinical cases. This study aims to identify and characterize an A. altamirensis isolate from chicken guts collected in an Italian slaughterhouse. The study approach includes whole-genome analysis jointly with phenotypical tests. Whole-genome sequencing (WGS) confirms the initial MALDI-TOF MS identification, finding putative virulence and biofilm-related genes. Moreover, the gene class evaluation reveals that the numerically largest gene category in the A. altamirensis genome is related to amino acid metabolism and transport. The analyses performed on a human gut mucus-producing cell line (HT29-MTX-E12) demonstrated the ability of A. altamirensis to colonize the host cell layer. Moreover, the antibiotic resistance test showed a high resistance of A. altamirensis to gentamicin (MIC 0.5 mg/L). The detection of a potential pathogenic and antibiotic-resistant A. altamirensis strain isolated from a slaughterhouse underlines the necessity of active surveillance studies focused on this species and the need for further studies about A. altamirensis in foods.

LAP-MALDI MS Profiling and Identification of Potential Biomarkers for the Detection of Bovine Tuberculosis.

Detecting bovine tuberculosis (bTB) primarily relies on the tuberculin skin test, requiring two separate animal handling events with a period of incubation time (normally 3 days) between them. Here, we present the use of liquid atmospheric pressure (LAP)-MALDI for the identification of bTB infection, employing a three-class prediction model that was obtained by supervised linear discriminant analysis (LDA) and tested with bovine mastitis samples as disease-positive controls. Noninvasive collection of nasal swabs was used to collect samples, which were subsequently subjected to a short (<4 h) sample preparation method. Cross-validation of the three-class LDA model from the processed nasal swabs provided a sensitivity of 75.0% and specificity of 90.1%, with an overall classification accuracy of 85.7%. These values are comparable to those for the skin test, showing that LAP-MALDI MS has the potential to provide an alternative single-visit diagnostic platform that can detect bTB within the same day of sampling.

A comparative study for optimization of MALDI-TOF MS identification of filamentous fungi.

To evaluate and compare the performance of three commercial culture media, two filamentous fungi libraries, and two different protein extraction procedures in MALDI-TOF MS fungal identification. A total of 21 quality control samples were cultured on Sabouraud dextrose agar (SDA), ID fungi plate medium (IDFP), and Sabouraud gentamicin chloramphenicol 2 agar (SGC2). For four consecutive days, fungal growths were inoculated on a MALDI target plate both by using a direct transfer technique (DT) and by using a formic acid-ethanol protein extraction procedure (EEP). The MALDI-TOF MS-generated spectra were identified by the MBT Bruker library and the MSI database. Selective culture media (IDFP and SGC2) significantly outperformed the non-selective SDA medium. IDFP was superior to the SGC2 medium for dermatophyte identification. The EEP only demonstrated a benefit over DT in the underperforming SDA medium. The MBT Bruker library outperformed the MSI database in Aspergillus identification while the MSI database outperformed the MBT library in dermatophyte identification. For non-Aspergillus fungi, the libraries performed comparably. The results of our study show the necessity of using selective culture media (IDFP and SGC2) for fungal identification with MALDI-TOF MS and demonstrate no significant benefit of the formic acid-ethanol protein extraction technique in these media. Given the relative strengths and weaknesses of the MBT library and the MSI database, it might currently be beneficial to consider these libraries as complementary and employ both databases to achieve optimal fungal identification.

Critical evaluation of MS acquisition conditions and identification process in LC-Q-Orbitrap-MS for non-targeted analysis: pesticide residues as case of study.

In routine measurements, the length of analysis time and the number of samples analysed during a given time unit are crucial. Additionally, the analytical method used has to provide reliable results and be able to identify and quantify any compound present in the matrix. High-resolution equipment, including Orbitrap analysers, is commonly used for non-targeted determinations. However, researchers still rely on trial and error to achieve the best acquisition conditions on the mass spectrometer, which is a tedious and time-consuming process that can lead to errors. Moreover, tentative compound identification, particularly when using a non-targeted approach, heavily depends on commercial databases. All of these issues can ultimately result in incomplete identification of compounds in the study matrix. In this framework, the study presented here has a dual objective: to use the experimental design tool to optimise critical parameters in mass spectrometry using LC-Q-Orbitrap-MS equipment when working in a non-targeted approach and to compare the mzCloud™ and ChemSpider™ commercial databases included in Compound Discoverer software with TraceFinder home-made databases generated to evaluate the ability to identify compounds. The study's noteworthy findings reveal that employing an experimental design has facilitated rapid optimisation of the mass spectrometer's multiplexing and loop parameters. Furthermore, the study highlights that the lack of harmonisation in commercial databases poses a disadvantage in the identification of compounds, leading to superior results when using home-made databases. In the latter databases, around 80% of the compounds were identified, which is approximately twice the number identified in commercial databases (around 40% in the best case with ChemSpider™).

Evaluation of the CHROMagar Candida Plus medium for presumptive identification of yeasts and MALDI-TOF MS identification.

Rapid and accurate yeasts species identification in clinical laboratories is important for appropriate and timely antifungal treatment. We evaluate the performance of the new medium CHROMagar™ Candida Plus for presumptive identification of yeasts species and MALDI-TOF identification. We identify 303 strains belonging to 60 clinically relevant yeasts species by using the new medium. Presumptive identification was correct at the Candida albicans complex, Candida tropicalis and Pichia kudriavzevii (Candida krusei) species. However, although this medium was able to identify all Candida auris and Candida glabrata strains, other species were misidentified as C. auris or C. glabrata. A total of 215 strains were identified by using MALDI-TOF and evaluated two incubation temperatures (30°C and 37°C) and two incubation times (24 h and 72 h). Most strains (94%; 202/215) were correctly identified at the species (n:190) or complex level (n:12) at both temperatures and incubation times. However, we observed that the time of incubation (24 h vs. 72 h) affects the score values when yeasts are incubated at 37°C, but does not affect score values when yeasts are incubated at 30°C. In conclusion, the new medium has a good performance in the presumptive identification of the C. albicans complex, C. tropicalis and P. kudriavzevii (C. krusei). In addition, this medium is useful for the screening of C. auris and C. glabrata isolates, but identification should be confirmed by other more specific techniques, like MALDI-TOF.

Improved MALDI-TOF MS Identification of Mycobacterium tuberculosis by Use of an Enhanced Cell Disruption Protocol.

Mycobacterium tuberculosis is the microorganism that causes tuberculosis, a disease affecting millions of people worldwide. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable, and cost-effective method for microorganism identification which has been used for the identification of Mycobacterium spp. isolates. However, the mycobacteria cell wall is rich in lipids, which makes it difficult to obtain proteins for MALDI-TOF MS analysis. In this study, two cell preparation protocols were compared: the MycoEx, recommended by MALDI-TOF instrument manufacturer Bruker Daltonics, and the MycoLyser protocol described herein, which used the MagNA Lyser instrument to enhance cell disruption with ethanol. Cell disruption and protein extraction steps with the two protocols were performed using the Mycobacterium tuberculosis H37Rv strain, and the MALDI-TOF MS results were compared. The MycoLyser protocol allowed for improved Biotyper identification of M. tuberculosis since the log(score) values obtained with this protocol were mostly ≥ 1.800 and significantly higher than that underwent MycoEx processing. The identification reliability was increased as well, considering the Bruker criteria. In view of these results, it is concluded that the MycoLyser protocol for mycobacterial cell disruption and protein extraction improves the MALDI-TOF MS method's efficacy for M. tuberculosis identification.

MALDI-TOF MS identification of Exophiala species isolated in Japan: Library enrichment and faster sample preparation.

Exophiala species cause chromoblastomycosis, mycetoma, and phaeohyphomycosis, which are occasionally fatally in immunocompromised patients. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides rapid and accurate examination of isolated bacteria and some fungal isolates, but the preparation method for filamentous fungi is complicated. In this study, 31 clinical isolates of Exophiala spp. in Japan were identified by MALDI-TOF MS with a library enriched by adding data. To simplify the sample preparation method, two modified methods were compared with the standard method for filamentous fungi. The agar cultivation sample preparation method reduced the time required for liquid culture and was considered suitable for clinical use. In 30 of 31 clinical isolates of Exophiala spp., the species identified by MALDI-TOF MS with the highest score matched the species identified by sequencing the internal transcribed spacer region. Exophiala dermatitidis, E. lecanii-corni, and E. oligosperma were identified above the genus level, while E. jeanselmei and E. xenobiotica were often not identified at the species level. The identification scores tended to be lower for less-registered strains in the in-house library. It is suggested that library enrichment and the modified preparation method may facilitate early diagnosis of rare fungal infections by Exophiala spp. in clinical laboratories using MALDI-TOF MS.

Plant-growth promoting activity of three fungal endophytes isolated from plants living in dehesas and their effect on Lolium multiflorum

Endophytic fungi have been demonstrated to produce bioactive secondary metabolites, some of which promote plant growth. Three endophytic fungi isolated from healthy plants living in dehesas of Extremadura (Spain) were identified and evaluated for their ability to produce phytohormone-like substances, antioxidant activity, total polyphenol content, phosphate solubilization ability and siderophore and ammonia production. The filtrates and extracts produced by the three endophytes were applied to Lolium multiflorum seeds and seedlings under both in vitro and greenhouse conditions, to analyse their influence on plant growth traits such as germination, vigour index, chlorophyll data, number and length of leaves and roots, and dry weight. All three endophytes, which were identified as Fusarium avenaceum, Sarocladium terricola and Xylariaceae sp., increased the germination of L. multiflorum seeds by more than 70%. Shoot and root length, plant dry weight and the number of roots were positively affected by the application of fungal filtrates and/or extracts, compared with controls. The tentative HPLC–MS identification of phytohormone-like substances, such as gibberellin A2 and zeatin, or the antioxidant acetyl eugenol, may partially explain the mechanisms of L. multiflorum plant growth promotion after the application of fungal filtrates and/or extracts.

MALDI-TOF MS Is an Effective Technique To Classify Specific Microbiota.

MALDI-TOF MS is well-recognized for single microbial identification and widely used in research and clinical fields due to its specificity, speed of analysis, and low cost of consumables. Multiple commercial platforms have been developed and approved by the U.S. Food and Drug Administration. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used for microbial identification. However, microbes can present as a specific microbiota, and detection and classification remain a challenge. Here, we constructed several specific microbiotas and tried to classify them using MALDI-TOF MS. Different concentrations of nine bacterial strains (belonging to eight genera) constituted 20 specific microbiotas. Using MALDI-TOF MS, the overlap spectrum of each microbiota (MS spectra of nine bacterial strains with component percentages) could be classified by hierarchical clustering analysis (HCA). However, the real MS spectrum of a specific microbiota was different than that of the overlap spectrum of component bacteria. The MS spectra of specific microbiota showed excellent repeatability and were easier to classify by HCA, with an accuracy close to 90%. These results indicate that the widely used MALDI-TOF MS identification method for individual bacteria can be expanded to classification of microbiota. IMPORTANCE MALDI-TOF MS can be used to classify specific model microbiota. The actual MS spectrum of the model microbiota was not a simple superposition of every single bacterium in a certain proportion but had a specific spectral fingerprint. The specificity of this fingerprint can enhance the accuracy of microbiota classification.

Authentication of Single Herbal Powders Enabled by Microscopy-Guided In Situ Auto-sampling Combined with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

In the long history of investigation of herbal products, microscopic examination has greatly contributed to the authentication of herbs in a powder form. However, it cannot provide the chemical profiles of herbal powders and thus is limited to morphological identification. In this work, we present a label-free and automatic approach for the characterization and identification of single herbal powders and their adulterants, enabled through the combination of microscopy-guided auto-sampling and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). To meet the demand for automatic and highly efficient in situ extraction, the glass slide was coated with gelatin to immobilize dried herbal powders that cannot stick to the glass slide like fresh and hydrated cells. The gelatin coating also facilitated the pump-out of chemical components and prevented diffusion across the interface enabled by the formation of a tight contact at the probe tip and surface. Optical microscopy was applied to acquire the microstructure and position of the herbal powders immobilized on the gelatin-coated slide. The candidate single herbal powders were picked out by a software for subsequent auto-sampling and MALDI MS identification. The combination of microstructure features and chemical profiles significantly improved the authentication capability of microscopic examination.