A Comprehensive Comparison of Tissue Processing Methods for High-Quality MALDI Imaging of Lipids in Reconstructed Human Epidermis.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has become an important tool for skin analysis, as it allows the simultaneous detection and localization of diverse molecular species within a sample. The use of in vivo and ex vivo human skin models is costly and presents ethical issues; therefore, reconstructed human epidermis (RHE) models, which mimic the upper part of native human skin, represent a suitable alternative to investigate adverse effects of chemicals applied to the skin. However, there are few publications investigating the feasibility of using MALDI MSI on RHE models. Therefore, the aim of this study was to investigate the effect of sample preparation techniques, i.e., substrate, sample thickness, washing, and matrix recrystallization, on the quality of MALDI MSI for lipids analysis of the SkinEthic RHE model. Images were generated using an atmospheric pressure MALDI source coupled to a high-resolution mass spectrometer with a pixel size of 5 µm. Masses detected in a defined region of interest were analyzed and annotated using the LipostarMSI platform. The results indicated that the combination of (1) coated metallic substrates, such as APTES-coated stainless-steel plates, (2) tissue sections of 6 µm thickness, and (3) aqueous washing before HCCA matrix spraying (without recrystallization), resulted in images with a significant signal intensity as well as numerous m/z values. This refined methodology using AP-MALDI coupled to a high-resolution mass spectrometer should improve the current sample preparation workflow to evaluate changes in skin composition after application of dermatocosmetics.

Formate promotes invasion and metastasis in reliance on lipid metabolism.

Metabolic rewiring is essential for cancer onset and progression. We previously showed that one-carbon metabolism-dependent formate production often exceeds the anabolic demand of cancer cells, resulting in formate overflow. Furthermore, we showed that increased extracellular formate concentrations promote the in vitro invasiveness of glioblastoma cells. Here, we substantiate these initial observations with ex vivo and in vivo experiments. We also show that exposure to exogeneous formate can prime cancer cells toward a pro-invasive phenotype leading to increased metastasis formation in vivo. Our results suggest that the increased local formate concentration within the tumor microenvironment can be one factor to promote metastases. Additionally, we describe a mechanistic interplay between formate-dependent increased invasiveness and adaptations of lipid metabolism and matrix metalloproteinase activity. Our findings consolidate the role of formate as pro-invasive metabolite and warrant further research to better understand the interplay between formate and lipid metabolism.

MALDI Mass Spectrometry Imaging: A Potential Game-Changer in a Modern Microbiology.

Nowadays, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is routinely implemented as the reference method for the swift and straightforward identification of microorganisms. However, this method is not flawless and there is a need to upgrade the current methodology in order to free the routine lab from incubation time and shift from a culture-dependent to an even faster independent culture system. Over the last two decades, mass spectrometry imaging (MSI) gained tremendous popularity in life sciences, including microbiology, due to its ability to simultaneously detect biomolecules, as well as their spatial distribution, in complex samples. Through this literature review, we summarize the latest applications of MALDI-MSI in microbiology. In addition, we discuss the challenges and avenues of exploration for applying MSI to solve current MALDI-TOF MS limits in routine and research laboratories.

Investigation of MALDI-TOF Mass Spectrometry for Assessing the Molecular Diversity of Campylobacter jejuni and Comparison with MLST and cgMLST: A Luxembourg One-Health Study.

There is a need for active molecular surveillance of human and veterinary Campylobacter infections. However, sequencing of all isolates is associated with high costs and a considerable workload. Thus, there is a need for a straightforward complementary tool to prioritize isolates to sequence. In this study, we proposed to investigate the ability of MALDI-TOF MS to pre-screen C. jejuni genetic diversity in comparison to MLST and cgMLST. A panel of 126 isolates, with 10 clonal complexes (CC), 21 sequence types (ST) and 42 different complex types (CT) determined by the SeqSphere+ cgMLST, were analysed by a MALDI Biotyper, resulting into one average spectra per isolate. Concordance and discriminating ability were evaluated based on protein profiles and different cut-offs. A random forest algorithm was trained to predict STs. With a 94% similarity cut-off, an AWC of 1.000, 0.933 and 0.851 was obtained for MLSTCC, MLSTST and cgMLST profile, respectively. The random forest classifier showed a sensitivity and specificity up to 97.5% to predict four different STs. Protein profiles allowed to predict C. jejuni CCs, STs and CTs at 100%, 93% and 85%, respectively. Machine learning and MALDI-TOF MS could be a fast and inexpensive complementary tool to give an early signal of recurrent C. jejuni on a routine basis.

Evaluating Different Storage Media for Identification of Taenia saginata Proglottids Using MALDI-TOF Mass Spectrometry.

Taenia saginata is a helminth that can cause taeniasis in humans and cysticercosis in cattle. A species-specific diagnosis and differentiation from related species (e.g., Taenia solium) is crucial for individual patient management and disease control programs. Diagnostic stool microscopy is limited by low sensitivity and does not allow discrimination between T. saginata and T. solium. Molecular diagnostic approaches are not routinely available outside research laboratories. Recently, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was proposed as a potentially suitable technique for species-specific helminth diagnosis. However, standardized protocols and commercial databases for parasite identification are currently unavailable, and pre-analytical factors have not yet been assessed. The purpose of this study was to employ MALDI-TOF MS for the identification of T. saginata proglottids obtained from a human patient, and to assess the effects of different sample storage media on the technique's diagnostic accuracy. We generated T. saginata-specific main spectral profiles and added them to an in-house database for MALDI-TOF MS-based diagnosis of different helminths. Based on protein spectra, T. saginata proglottids could be successfully differentiated from other helminths, as well as bacteria and fungi. Additionally, we analyzed T. saginata proglottids stored in (i) LC-MS grade water; (ii) 0.45% sodium chloride; (iii) 70% ethanol; and (iv) 37% formalin after 2, 4, 6, 8, 12, and 24 weeks of storage. MALDI-TOF MS correctly identified 97.2-99.7% of samples stored in water, sodium chloride, and ethanol, with log-score values ≥2.5, thus indicating reliable species identification. In contrast, no protein spectra were obtained for samples stored in formalin. We conclude that MALDI-TOF-MS can be successfully employed for the identification of T. saginata, and that water, sodium chloride, and ethanol are equally effective storage solutions for prolonged periods of at least 24 weeks.

Combination of MALDI-TOF Mass Spectrometry and Machine Learning for Rapid Antimicrobial Resistance Screening: The Case of Campylobacter spp.

While MALDI-TOF mass spectrometry (MS) is widely considered as the reference method for the rapid and inexpensive identification of microorganisms in routine laboratories, less attention has been addressed to its ability for detection of antimicrobial resistance (AMR). Recently, some studies assessed its potential application together with machine learning for the detection of AMR in clinical pathogens. The scope of this study was to investigate MALDI-TOF MS protein mass spectra combined with a prediction approach as an AMR screening tool for relevant foodborne pathogens, such as Campylobacter coli and Campylobacter jejuni. A One-Health panel of 224 C. jejuni and 116 C. coli strains was phenotypically tested for seven antimicrobial resistances, i.e., ciprofloxacin, erythromycin, tetracycline, gentamycin, kanamycin, streptomycin, and ampicillin, independently, and were submitted, after an on- and off-plate protein extraction, to MALDI Biotyper analysis, which yielded one average spectra per isolate and type of extraction. Overall, high performance was observed for classifiers detecting susceptible as well as ciprofloxacin- and tetracycline-resistant isolates. A maximum sensitivity and a precision of 92.3 and 81.2%, respectively, were reached. No significant prediction performance differences were observed between on- and off-plate types of protein extractions. Finally, three putative AMR biomarkers for fluoroquinolones, tetracyclines, and aminoglycosides were identified during the current study. Combination of MALDI-TOF MS and machine learning could be an efficient and inexpensive tool to swiftly screen certain AMR in foodborne pathogens, which may enable a rapid initiation of a precise, targeted antibiotic treatment.

MALDI-TOF Mass Spectrometry and Specific Biomarkers: Potential New Key for Swift Identification of Antimicrobial Resistance in Foodborne Pathogens.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is today the reference method for direct identification of microorganisms in diagnostic laboratories, as it is notably time- and cost-efficient. In the context of increasing cases of enteric diseases with emerging multi-drug resistance patterns, there is an urgent need to adopt an efficient workflow to characterize antimicrobial resistance (AMR). Current approaches, such as antibiograms, are time-consuming and directly impact the "patient-physician" workflow. Through this mini-review, we summarize how the detection of specific patterns by MALDI-TOF MS, as well as bioinformatics, become more and more essential in research, and how these approaches will help diagnostics in the future. Along the same lines, the idea to export more precise biomarker identification steps by MALDI-TOF(/TOF) MS data towards AMR identification pipelines is discussed. The study also critically points out that there is currently still a lack of research data and knowledge on different foodborne pathogens as well as several antibiotics families such as macrolides and quinolones, and many questions are still remaining. Finally, the innovative combination of whole-genome sequencing and MALDI-TOF MS could be soon the future for diagnosis of antimicrobial resistance in foodborne pathogens.

MALDI-TOF mass spectrometry as a diagnostic tool in human and veterinary helminthology: a systematic review.

BACKGROUND: Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a widely used technique for the rapid and accurate identification of bacteria, mycobacteria and certain fungal pathogens in the clinical microbiology laboratory. Thus far, only few attempts have been made to apply the technique in clinical parasitology, particularly regarding helminth identification. METHODS: We systematically reviewed the scientific literature on studies pertaining to MALDI-TOF MS as a diagnostic technique for helminths (cestodes, nematodes and trematodes) of medical and veterinary importance. Readily available electronic databases (i.e. PubMed/MEDLINE, ScienceDirect, Cochrane Library, Web of Science and Google Scholar) were searched from inception to 10 October 2018, without restriction on year of publication or language. The titles and abstracts of studies were screened for eligibility by two independent reviewers. Relevant articles were read in full and included in the systematic review. RESULTS: A total of 84 peer-reviewed articles were considered for the final analysis. Most papers reported on the application of MALDI-TOF for the study of Caenorhabditis elegans, and the technique was primarily used for identification of specific proteins rather than entire pathogens. Since 2015, a small number of studies documented the successful use of MALDI-TOF MS for species-specific identification of nematodes of human and veterinary importance, such as Trichinella spp. and Dirofilaria spp. However, the quality of available data and the number of examined helminth samples was low. CONCLUSIONS: Data on the use of MALDI-TOF MS for the diagnosis of helminths are scarce, but recent evidence suggests a potential role for a reliable identification of nematodes. Future research should explore the diagnostic accuracy of MALDI-TOF MS for identification of (i) adult helminths, larvae and eggs shed in faecal samples; and (ii) helminth-related proteins that are detectable in serum or body fluids of infected individuals.