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Site-to-Site Reproducibility and Spatial Resolution in MALDI-MSI of Peptides from Formalin-Fixed Paraffin-Embedded Samples
摘要： Purpose To facilitate the transition of MALDI-MS Imaging (MALDI-MSI) from basic science to clinical application, it is necessary to analyze formalin-fixed paraffin-embedded (FFPE) tissues. Our aim was to improve in-situ tryptic digestion for MALDI-MSI of FFPE samples and determine if similar results would be reproducible if obtained from different sites. Experimental Design FFPE tissues (mouse intestine, human ovarian teratoma, tissue microarray of tumor entities sampled from three different sites) were prepared for MALDI-MSI. Samples were coated with trypsin using an automated sprayer then incubated using deliquescence to maintain a stable humid environment. After digestion, samples were sprayed with CHCA using the same spraying device and analyzed with a rapifleX MALDI Tissuetyper at 50μm spatial resolution. Data were analyzed using flexImaging, SCiLS and R. Results Trypsin application and digestion were identified as sources of variation and loss of spatial resolution in the MALDI-MSI of FFPE samples. Using the described workflow, it is possible to discriminate discrete histological features in different tissues and enabled different sites to generate images of similar quality when assessed by spatial segmentation and PCA. Conclusions and Clinical Relevance Spatial resolution and site-to-site reproducibility can be maintained by adhering to a standardized MALDI-MSI workflow.
关键词： Reproducibility,Tissue typing,MALDI,Workflow,Formalin-fixed paraffin embedded tissue
更新于2025-09-23 15:21:21
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New Derivatization Reagent for Detection of free Thiol-groups in Metabolites and Proteins in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging
摘要： Several diseases are associated with disturbed redox signaling and altered metabolism of sulfur-containing metabolites and proteins. Importantly, oxidative degradation of fresh-frozen tissues begins within the normal timescale of MALDI MSI sample preparation. As a result, analytical methods that preserve the redox state of the tissue are urgently needed for refined studies of the underlying mechanisms. Nevertheless, no derivatization strategy for free sulfhydryl groups in tissue is known for MALDI MSI. Here, we report the first derivatization reagent, (E)-2-cyano-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-(4-hydroxyphenyl)acrylamide (CHC-Mal), for selective detection of free thiols using MALDI MSI. We performed in-situ derivatization of free thiol groups from thiol-containing metabolites such as glutathione and cysteine and reduced proteins such as insulin and imaged their spatial distribution in porcine and mouse xenograft tissue. Derivatization of thiol-containing metabolites with CHC-Mal for MALDI MSI was also possible when using aged tissue in the presence of excess reducing agents. Importantly, CHC-Mal-derivatized low mass-metabolites could be detected without the use of a conventional MALDI matrix.
关键词： redox signaling,sulfur-containing metabolites,MALDI MSI,derivatization reagent,free thiol-groups
更新于2025-09-23 15:21:01
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Plasmonic Colloidosomes Coupled MALDI-TOF MS for Bacterial Heteroresistance Study at Single-Cell Level
摘要： Antimicrobial resistance (AMR) is a long-term public health challenge worldwide, and it is increasingly recognized to be a heteroresistance phenomenon in an isogenic bacterial population. When the minority population of resistant bacteria with strong AMR is not handled in time, such sub-population can be enriched leading to the further development of bacterial AMR. However, conventional AMR studies based on ensemble-averaged data from large population fail to characterize the bacterial heterogeneity. In this work, we develop a method using plasmonic colloidosomes and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to study single bacterial cell AMR. The plasmonic colloidosomes act simultaneously as bacteria containers and sample spots for MALDI-TOF MS detection. Cells of β-lactamases producing Escherichia coli (E. coli) are trapped in colloidosome containers (~200 μm in diameter) in the presence of antibiotic drug ampicillin (AMP). Benefiting from the fast reaction kinetics in microcompartments, hydrolysis product of AMP by bacteria can be detected by MALDI-TOF MS within 40 min. The colloidosomes as MALDI sample spots also benefit sensitive detection and accurate quantification of AMP and its hydrolysis product. It was found that even an isogenic population could consist of a mixture of bacteria that have different resistance degrees to antibiotics. Taken the β-lactamases producing E. coli as an example, 20% of the bacterial individuals have relatively strong activity in hydrolyzing AMP. It is expected that the colloidosome-based platform would reveal a prospective application in full characterization of single bacterial cell AMR.
关键词： MALDI-TOF MS,Antimicrobial resistance,Single-cell level,Plasmonic colloidosomes,Bacterial heteroresistance
更新于2025-09-23 15:21:01
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High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
摘要： Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.
关键词： bacteria,metabolic fingerprinting,matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS),barium monocarboxylates,free fatty acids (FFAs),chemical deposition technique,Langmuir film technology,rhizobia
更新于2025-09-23 15:21:01
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An approach for broad molecular imaging of the root-soil interface via indirect matrix-assisted laser desorption/ionization mass spectrometry
摘要： Understanding rhizospheric processes is limited by the need for imaging complex molecular transformations at relevant spatial scales within the root soil continuum. Here, we demonstrate a method to enable this analysis by first extracting organic compounds from the rhizosphere onto a PVDF membrane while maintaining their 2D distribution and then imaging the distribution of chemical compounds using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This approach permitted us to visualize and identify compounds on the root surface and presumed root exudates in the rhizosphere. Within a 1.8 cm x 0.6 cm sampling area of a switchgrass rhizosphere, we could observe at least four chemically distinct zones. Using high performance Fourier transform ion cyclotron MS, we were able to accurately annotate numerous molecules co-localized to each of these zones.
关键词： chemical imaging,PVDF membrane,spatial metabolomics,MALDI,rhizosphere,liquid chromatography
更新于2025-09-23 15:21:01
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Supervised Non-negative Matrix Factorization Methods for MALDI Imaging Applications
摘要： Motivation: Non-negative matrix factorization (NMF) is a common tool for obtaining low-rank approximations of non-negative data matrices and has been widely used in machine learning, e.g., for supporting feature extraction in high-dimensional classification tasks. In its classical form NMF is an unsupervised method, i.e. the class labels of the training data are not used when computing the NMF. However, incorporating the classification labels into the NMF algorithms allows to specifically guide them towards the extraction of data patterns relevant for discriminating the respective classes. This approach is particularly suited for the analysis of mass spectrometry imaging (MSI) data in clinical applications, such as tumor typing and classification, which are amongst the most challenging tasks in pathology. Thus, we investigate algorithms for extracting tumor specific spectral patterns from MSI data by NMF methods. Results: In this paper, we incorporate a priori class labels into the NMF cost functional by adding appropriate supervised penalty terms. Numerical experiments on a MALDI imaging dataset confirm that the novel supervised NMF methods lead to significantly better classification accuracy and stability as compared to other standard approaches.
关键词： MALDI imaging,tumor typing,Non-negative matrix factorization,mass spectrometry imaging,supervised learning
更新于2025-09-23 15:21:01
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MALDI MS Imaging at Acquisition Rates Exceeding 100 Pixels per Second
摘要： The practicality of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) applied to molecular imaging of biological tissues is limited by the analysis speed. Typically, a relatively low speed of stop-and-go micromotion of XY stages is considered as a factor substantially reducing the rate with which fresh sample material can be supplied to the laser spot. The sample scan rate in our laboratory-built high-throughput imaging TOF mass spectrometer was significantly improved through the use of a galvanometer-based optical scanner performing fast laser spot repositioning on a target plate. The optical system incorporated into the ion source of our MALDI TOF mass spectrometer allowed focusing the laser beam via a modified grid into a 10-μm round spot. This permitted the acquisition of high-resolution MS images with a well-defined pixel size at acquisition rates exceeding 100 pixel/s. The influence of selected parameters on the total MS imaging time is discussed. The new scanning technique was employed to display the distribution of an antitumor agent in 3D colorectal adenocarcinoma cell aggregates; a single MS image comprising 100 × 100 pixels with 10-μm lateral resolution was recorded in approximately 70 s.
关键词： MSI,TOF,Mass spectrometry imaging,High throughput,Laser beam scanning,Colorectal adenocarcinoma,3D cell aggregates,Spheroids,Grid ion source,MALDI
更新于2025-09-23 15:21:01
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Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for accurate discrimination of Staphylococcus schleiferi subspecies
摘要： S. schleiferi is one of the main species isolated from canine otitis externa, pyoderma and from apparently healthy dogs. The species is divided into two subspecies, S. schleiferi schleiferi and S. schleiferi coagulans. MALDI-TOF MS does not distinguish correctly these two subspecies. This study aimed to identify biomarkers that could possibly discriminate Staphylococcus schleiferi subspecies by MALDI-TOF MS. Twelve strains (eight S. schleiferi schleiferi and four S. schleiferi coagulans) were firstly identified. Each isolate was submitted to a protein extraction protocol and used for improved subspecies identification in clinical samples.
关键词： MALDI-TOF MS,Staphylococcus schleiferi,biomarkers
更新于2025-09-23 15:19:57
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Metabolomic approach for rapid differentiation of Fritillaria bulbs by matrix-assisted laser desorption/ionization mass spectrometry and multivariate statistical analysis
摘要： The bulbs of Fritillaria have been used for centuries as food and medicinal products in many Asian countries. Different Fritillaria species have distinct pharmacological effects despite of their similar appearances. Effective differentiation of Fritillaria species can avoid adulteration and is crucial to its clinical uses. In this paper, a hybrid method of matrix assisted laser desorption/ionization mass spectrometry and multivariate statistical analysis was developed for the rapid and reliable differentiation of Fritillaria species for the first time. Significantly different patterns for five Fritillaria species were obtained by MALDI-MS after instant sample extractions. Different groups of Fritillaria were confidently differentiated via an orthogonal partial least square model. In addition, a metabolomic taxonomy of five Fritillaria species was obtained based on MALDI-MS data.
关键词： Fritillaria,metabolic profiling,MALDI-MS,multivariate statistical analysis,chemical taxonomy
更新于2025-09-23 15:19:57
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Rapid quality control of medicine and food dual purpose plant polysaccharides by matrix assisted laser desorption/ionization mass spectrometry
摘要： With the multiple biological activities and health benefit effects, polysaccharide in medicine and food dual purpose plants (MFDPPP) has been extensively applied in many fields, such as medical treatment, stock farming, and cosmetic industry. However, up to date, quality issues of MFDPPP and analytical technologies of polysaccharide post challenges to chemists. Reported herein is a rapid and high-throughput quality control method for analyzing MFDPPP, based on the matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). For the analysis of illegal addition and doping, ferroferric oxide nanoparticles was screened out as MALDI matrix to avoid the small molecule interference. Qualitatively, high sensitivity was obtained for both illegal drugs and glucose. Quantitatively, the best linear response (R2 > 0.99) was attained at the concentration range from 0.005 to 1 mg·mL-1 for glucose as an example. For the analysis of polysaccharides, 2,5-dihydroxybenzoic acid/N-methylaniline was employed as MALDI matrix to increase the detection sensitivity and mass range coverage. Furthermore, the established method was successfully applied to the analysis of supplements from Astragalus polysaccharides and Lentinan real samples, respectively, thus showing its potential in quality control for MFDPPP.
关键词： medicine and food dual purpose plants,MALDI-MS,illegal adulterations,quality control,polysaccharides
更新于2025-09-23 15:19:57