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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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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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Quantitative of progesterone using isotope dilution-matrix-assisted laser desorption ionization-time of flight mass spectrometry
摘要： A quantification assay based on isotope dilution mass spectrometry to determine the concentration of progesterone in human serum was reported. Incorporated with 13C3-progesterone, serum samples were subjected to progesterone extraction and clean-up by C4 solid-phase-extraction columns and hexane-based liquid/liquid extraction, respectively. The cleaned-up serum samples were then subjected to MALDI-TOF mass spectrometry for the quantification of progesterone. In the study, the recovered progesterone concentration determined by the assay showed good robustness and constancy in comparison to conventional radioimmunologic assay. We concluded that the 13C3-progesterone-based quantification assay is a robust method for the measurement of serum progesterone.
关键词： Isotope dilution,MALDI-TOF/MS,Progesterone
更新于2025-09-23 15:19:57
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Investigation of the post mortem zinc protoporphyrin IX fluorescence with respect to its protein-bound and unbound occurrence in aqueous meat extracts
摘要： Zinc protoporphyrin IX (ZnPP) is known to accumulate in most meat products during storage. However, the pathway of its formation is not yet completely clarified. To gain more insights into the specificity of ZnPP occurrence, a SEC-HPLC-UV-fluorescence setup was established to screen the proteins in aqueous meat extracts for their ZnPP fluorescence during incubation. In accordance with previous studies it was identified by SDS-PAGE and MALDI-TOF-MS that ZnPP formation takes place in myoglobin. In this study, valuable new insights into the ZnPP forming pathway were gained, as our results indicated that a significant part of ZnPP - after being formed within the protein - is transitioned into free ZnPP during incubation. Additionally, the obtained results implied that ZnPP may also occur in proteins of higher molecular weight (>100 kDa).
关键词： SEC-HPLC,SDS-PAGE,post mortem chemistry,MALDI-TOF-MS,meat,fluorescence screening
更新于2025-09-19 17:15:36
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3a??Hydroxypicolinic Acid as an Effective Matrix for Sophorolipid Structural Elucidation Using Matrixa??Assisted Laser Desorption Ionization Timea??ofa??Flight Mass Spectrometry
摘要： Determination of the distribution of sophorolipid (SL) congeners controls their self-assembly structures and therefore their employment as glycolipid biosurfactants in cosmetics, environmental remediation, and personal care applications. The compatibility of 3-hydroxypicolinic acid (HPA) as a matrix for compositional analysis of SL using palm olein as carbon energy source by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis under positive and negative ion modes is studied in this work. Examination in positive ion mode revealed ions that were not detected in negative ion mode. Molecular ions produced using HPA matrix were lactonic Glc-Glc-10,400-C18:1, anionic Glc-Glc-10-C18:1, lactonic monoacetate of Glc-Glc-10,400-C18:1, and anionic monoacetate Glc-Glc-10-C18:1 and the anionic diacetate of also detected Glc-Glc-10-C18:0, Glc-Glc-10-C18:1. We GlcOAc-Glc-10-C18:0, GlcOAc-GlcOAc-10,400-C18:0, and GlcOAc—GlcOAc-10-C18:0 which are congeners of SL with stearic acid moieties. 3-HPA has been established to be the superior matrix for elucidation of structures for SL using MALDI-TOF MS.
关键词： Biosurfactant,Palm oil,3-Hydroxypicolinic acid,MALDI-TOF MS,Sophorolipids
更新于2025-09-19 17:13:59
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Matrixa??assisted laser desorption/ionization behavior of neat linear and cyclic poly(La??lactide)s and their blends
摘要： Rationale Numerous new tin catalysts were recently developed that enable the synthesis of cyclic polylactides with broad variation of the molecular mass. The abundance of cyclics in MALDI TOF mass spectra is, however, frequently reported to greatly exceed that of linears. Thus the MALDI ionization behavior of various end-capped linear poly(L-lactide)s and one cyclic poly(L-lactide) was investigated and compared. Methods Neat compounds and various blends of cyclic and linear species were prepared and studied under identical conditions with regard to sample preparation and instrumental condition, except for the laser power. For this purpose, two different MALDI TOF mass spectrometers were applied. Results Our results reveal that cyclics indeed show a slightly better ionization in MALDI, although their ionization as neat compound seems to be less effective than that of linear polylactides. The ionization of most linear polylactides investigated does not depend on the end group structure. However, linear polylactides containing 12-bromododecyl end groups reveal an unexpected saturation effect that is not caused by fragmentation of the polymer or the end group, or by electronic saturation of the detector digitizer. Furthermore, polylactides with a 2- bromoethyl end group did not show such a saturation effect. Conclusions An overestimation of cyclic species in MALDI TOF mass spectra of poly(L-lactide)s must be considered, but the commonly assumed peak suppression of linear polymers in mixtures of both architectures can be excluded.
关键词： cyclic,poly(L-lactide),MALDI TOF MS,linear,abundance
更新于2025-09-19 17:13:59
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The Diagnosis of Sulfated Hemoglobin (SulfHb) Secondary to Sulfur Dioxide Poisoning Using Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-ToF MS)a??A Novel Approach to an Unusual Clinical Problem
摘要： Sulfhemoglobinemia is a rare entity caused by irreversible sulfation of the heme moiety in haemoglobin to form sulfated haemoglobin (SulfHb) and has been caused by H2S arising from certain metabolites of drugs and bacterial infection. Clinical presentation is similar to that of methemoglobin (MetHb). Furthermore, it is often difficult to distinguish between the diagnosis of SulfHb from MetHb in arterial blood gas analysers due to the broad overlap in the optical density (OD) absorption spectra—that of SulfHb swamping the more distinct OD absorption shift seen with MetHb. The presence of SulfHb was suspected in a 73-year-old lady with low oxygen saturation (SaO2 ~75%), central cyanosis, and normal arterial oxygen partial pressure (pO2 ~12 kPa). Repeated arterial blood gas analysis on different systems returned error messages for MetHb quantification. There was an improvement in oxygen saturation and cyanosis after an exchange transfusion. A full OD spectrophotometry (500–700 nm) of the patient’s whole blood was suggestive of the presence of SulfHb, with a minor peak absorption at 620 nm. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) was undertaken on whole blood samples from the patient pre- and post-transfusion, alongside normal controls. These demonstrated the presence of SulfHb in the patient’s blood, identifying sulfur, sulfur monoxide, and sulfur dioxide bound to the heme moiety. This gave vital identification as to the cause of Hb sulfation, which was distinct from that previously reported. Levels fell after the exchange transfusion and were completely eradicated after the correct source, an Epsom Salts constipation tonic, was identified. MALDI-ToF mass spectrometry is a new, rapid, specific, and sensitive diagnostic test for rare hematological syndromes such as SulfHb. In addition, it can identify the specific compounds bound to heme. Here, we provide useful diagnostic evidence as to the source of SulfHb, which was via SO2 rather than the previously described H2S.
关键词： SulfHb diagnosis,sulfated haemoglobin,mass spectrometry,MALDI-TOF MS,arterial blood gas analysers,oxygen saturation,MetHb
更新于2025-09-19 17:13:59
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Rapid classification of group B Streptococcus serotypes based on matrix-assisted laser desorption ionization-time of flight mass spectrometry and machine learning techniques
摘要： Background: Group B streptococcus (GBS) is an important pathogen that is responsible for invasive infections, including sepsis and meningitis. GBS serotyping is an essential means for the investigation of possible infection outbreaks and can identify possible sources of infection. Although it is possible to determine GBS serotypes by either immuno-serotyping or geno-serotyping, both traditional methods are time-consuming and labor-intensive. In recent years, the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported as an effective tool for the determination of GBS serotypes in a more rapid and accurate manner. Thus, this work aims to investigate GBS serotypes by incorporating machine learning techniques with MALDI-TOF MS to carry out the identification. Results: In this study, a total of 787 GBS isolates, obtained from three research and teaching hospitals, were analyzed by MALDI-TOF MS, and the serotype of the GBS was determined by a geno-serotyping experiment. The peaks of mass-to-charge ratios were regarded as the attributes to characterize the various serotypes of GBS. Machine learning algorithms, such as support vector machine (SVM) and random forest (RF), were then used to construct predictive models for the five different serotypes (Types Ia, Ib, III, V, and VI). After optimization of feature selection and model generation based on training datasets, the accuracies of the selected models attained 54.9–87.1% for various serotypes based on independent testing data. Specifically, for the major serotypes, namely type III and type VI, the accuracies were 73.9 and 70.4%, respectively. Conclusion: The proposed models have been adopted to implement a web-based tool (GBSTyper), which is now freely accessible at http://csb.cse.yzu.edu.tw/GBSTyper/, for providing efficient and effective detection of GBS serotypes based on a MALDI-TOF MS spectrum. Overall, this work has demonstrated that the combination of MALDI-TOF MS and machine intelligence could provide a practical means of clinical pathogen testing.
关键词： GBS,Machine learning,Serotypes,Group B streptococcus,MALDI-TOF-MS
更新于2025-09-16 10:30:52
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Incorporating Statistical Test and Machine Intelligence Into Strain Typing of Staphylococcus haemolyticus Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
摘要： Staphylococcus haemolyticus is one of the most significant coagulase-negative staphylococci, and it often causes severe infections. Rapid strain typing of pathogenic S. haemolyticus is indispensable in modern public health infectious disease control, infections to prevent further infectious facilitating the identification of the origin of outbreak. Rapid identification enables the effective control of pathogenic infections, which is tremendously beneficial to critically ill patients. However, the existing strain typing methods, such as multi-locus sequencing, are of relatively high cost and comparatively time-consuming. A practical method for the rapid strain typing of pathogens, suitable for routine use in clinics and hospitals, is still not available. Matrix-assisted laser desorption ionization-time of flight mass spectrometry combined with machine learning approaches is a promising method to carry out rapid strain typing. In this study, we developed a statistical test-based method to determine the reference spectrum when dealing with alignment of mass spectra datasets, and constructed machine learning-based classifiers for categorizing different strains of S. haemolyticus. The area under the receiver operating characteristic curve and accuracy of multi-class predictions were 0.848 and 0.866, respectively. Additionally, we employed a variety of statistical tests and feature-selection strategies to identify the discriminative peaks that can substantially contribute to strain typing. This study not only incorporates statistical test-based methods to manage the alignment of mass spectra datasets but also provides a practical means to accomplish rapid strain typing of S. haemolyticus.
关键词： MALDI-TOF MS,strain typing,Fisher’s exact test,Staphylococcus haemolyticus,machine learning
更新于2025-09-16 10:30:52