Integrating MALDI-TOF Mass Spectrometry with Machine Learning Techniques for Rapid Antimicrobial Resistance Screening of Foodborne Bacterial Pathogens.

Although MALDI-TOF mass spectrometry (MS) is considered as the gold standard for rapid and cost-effective identification of microorganisms in routine laboratory practices, its capability for antimicrobial resistance (AMR) detection has received limited focus. Nevertheless, recent studies explored the predictive performance of MALDI-TOF MS for detecting AMR in clinical pathogens when machine learning techniques are applied. This chapter describes a routine MALDI-TOF MS workflow for the rapid screening of AMR in foodborne pathogens, with Campylobacter spp. as a study model.

Identifying Putative Biomarkers of Foodborne Pathogens Using a Metabolomic Approach.

Metabolomics is the study of low molecular weight biochemical molecules (typically <1500 Da) in a defined biological organism or system. In case of food systems, the term "food metabolomics" is often used. Food metabolomics has been widely explored and applied in various fields including food analysis, food intake, food traceability, and food safety. Food safety applications focusing on the identification of pathogen-specific biomarkers have been promising. This chapter describes a nontargeted metabolite profiling workflow using gas chromatography coupled with mass spectrometry (GC-MS) for characterizing three globally important foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica, from selective enrichment liquid culture media. The workflow involves a detailed description of food spiking experiments followed by procedures for the extraction of polar metabolites from media, the analysis of the extracts using GC-MS, and finally chemometric data analysis using univariate and multivariate statistical tools to identify potential pathogen-specific biomarkers.

Direct analysis in real time mass spectrometry (DART-MS/MS) for rapid urine opioid detection in a clinical setting.

BACKGROUND AND AIMS: Current laboratory methods for opioid detection involve an initial screening with immunoassays which offers efficient but non-specific results and a subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation which offers accurate results but requires extensive sample preparation and turnaround time. Direct Analysis in Real Time (DART) tandem mass spectrometry is evaluated as an alternative approach for accurate opioid detection with efficient sample preparation and turnaround time. MATERIALS AND METHODS: DART-MS/MS was optimized by testing the method with varying temperatures, operation modes, extraction methods, hydrolysis times, and vortex times. The method was evaluated for 12 opioids by testing the analytical measurement range, percent carryover, precision studies, stability, and method-to-method comparison with LC-MS/MS. RESULTS: DART-MS/MS shows high sensitivity and specificity for the detection of 6-acetylmorphine, codeine, hydromorphone, oxymorphone, hydrocodone, naloxone, buprenorphine, norfentanyl, and fentanyl in urine samples. However, its performance was suboptimal for norbuprenorphine, morphine and oxycodone. CONCLUSION: In this proof-of-concept study, DART-MS/MS is evaluated for its rapid quantitative definitive testing of opioids drugs in urine. Further research is needed to expand its application to other areas of drug testing.

Development and Validation of a Proximity Labeling Fusion Protein Construct to Identify the Protein-Protein Interactions of Transcription Factors.

Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors.

Examination of primary aromatic amines content in polylactic acid straws and migration into food simulants using SERS with LC-MS.

Polylactic acid (PLA) straws hold eco-friendly potential; however, residual diisocyanates used to enhance the mechanical strength can generate carcinogenic primary aromatic amines (PAAs), posing health risks. Herein, we present a rapid, comprehensive strategy to detecting PAAs in 18 brands of food-grade PLA straws and assessing their migration into diverse food simulants. Surface-enhanced Raman spectroscopy was conducted to rapidly screen straws for PAAs. Subsequently, qualitative determination of migrating PAAs into various food simulants (4 % acetic acid, 10 % ethanol, 50 % ethanol) occurred at 70 °C for 2 h using liquid chromatography-mass spectrometry. Three PAAs including 4,4'-methylenedianiline, 2,4'-methylenedianiline, and 2,4-diaminotoluene were detected in all straws. Specifically, 2,4-diaminotoluene in 50 % ethanol exceeded specific migration limit of 2 µg/kg, raising safety concerns. Notably, PAAs migration to 10 % and 50 % ethanol surpassed that to 4 % acetic acid within a short 2-hour period. Moreover, PLA straws underwent varying degrees of shape changes before and after migration. Straws with poly(butylene succinate) resisted deformation compared to those without, indicating enhanced heat resistance, while poly(butyleneadipate-co-terephthalate) improved hydrolysis resistance. Importantly, swelling study unveiled swelling effect wasn't the primary factor contributing to the increased PAAs migration in ethanol food simulant, as there was no significant disparity in swelling degrees across different food simulants. FT-IR and DSC analysis revealed higher PAAs content in 50 % ethanol were due to highly concentrated polar ethanol disrupting hydrogen bonds and van der Waal forces holding PLA molecules together. Overall, minimizing contact between PLA straws and alcoholic foods is crucial to avoid potential safety risks posed by PAAs.

Rapid and accurate detection of cinnamon oil adulteration in perilla leaf oil using atmospheric solids analysis probe-mass spectrometry.

Perilla leaf oil (PLO) is a global premium vegetable oil with abundant nutrients and substantial economic value, rendering it susceptible to potential adulteration by unscrupulous entrepreneurs. The addition of cinnamon oil (CO) is one of the main adulteration avenues for illegal PLOs. In this study, new and real-time ambient mass spectrometric methods were developed to detect CO adulteration in PLO. First, atmospheric solids analysis probe tandem mass spectrometry combined with principal component analysis and principal component analysis-linear discriminant analysis was employed to differentiate between authentic and adulterated PLO. Then, a spectral library was established for the instantaneous matching of cinnamaldehyde in the samples. Finally, the results were verified using the SRM mode of ASAP-MS/MS. Within 3 min, the three methods successfully identified CO adulteration in PLO at concentrations as low as 5% v/v with 100% accuracy. The proposed strategy was successfully applied to the fraud detection of CO in PLO.

Atriplex hortensis var. 'rubra' extracts and purified amaranthin-type pigments reduce oxidative stress and inflammatory response in LPS-stimulated RAW264.7 cells.

The use of direct injection ion mobility mass spectrometry (DI-IM-MS) to detect and identify betacyanin pigments in A. hortensis 'rubra' extracts was explored for the first time, with results compared to conventional LC-MS/MS analysis. The anti-inflammatory activities of leaf and seed extracts, alongside purified amaranthin and celosianin pigments, were investigated using a model of lipopolysaccharide (LPS)-activated murine macrophages. Extracts and purified pigments significantly inhibited the production of prostaglandin E2 and NO by up to 90% and 70%, respectively, and reduced the expression of Il6, Il1b, Nos2, and Cox2. Leaf and seed extracts also decreased secretion of Il6 and Il1b cytokines and reduced protein levels of Nos2 and Cox2. Furthermore, extracts and purified pigments demonstrated potent dose-dependent radical scavenging activity in a cellular antioxidant activity assay (CAA) without any cytotoxic effects. Our research highlights the promising biological potential of edible, climate-resilient A. hortensis 'rubra' as a valuable source of bioactive compounds.

Seasonal changes of chemodiversity along with microbial succession in a municipal wastewater treatment plant.

The relationship between chemodiversity and microbial succession in wastewater treatment plants (WWTPs) is highly intricate and bidirectional. The specific contribution of the microbial community to changes in the composition of dissolved organic matter (DOM) within different biological treatment units remains unclear, as does the reciprocal influence of DOM composition on microbial succession. In this study, spectroscopy ((Excitation-emission matrix) EEM-PARAFAC, Ultraviolet (UV)-spectrum, Fourier transform infrared spectrometer (FT-IR)), Liquid chromatograph mass spectrometer (LC‒MS) and Fourier transform ion cyclotron resonance (FT-ICR) MS along with high-throughput sequencing technology were used to explore the relationship between chemodiversity and microbial succession in WWTPs concerning seasonal changes. The results showed that WWTPs with anaerobic/anoxic/oxic (A2O) processes can metabolize and transform most of the wastewater DOM, and the anaerobic unit has the highest removal rate for fluorescence DOM (FDOM, 14.07%-64.43%); the anaerobic unit increased aliphatic/proteins and lignin-like molecules but decreased relative intensity, while the anoxic unit removed unsaturated hydrocarbons, aromatic structures, and lignin-like substances. The impact of seasonal changes on the composition and removal of FDOM and DOM in wastewater treatment is significant, and the variations that occur during different seasons affect microbial activity, as well as the production, degradation, and transformation of organic compounds throughout the wastewater treatment process. Network analysis shows that Parcubacteria_genera_incertae_sedis plays a crucial role in DOM chemodiversity, highlighting the crucial contribution of microbial communities to both the structure and operation of the entire DOM network. The results in this study could provide some theoretical and practical basis for guiding the process optimization of WWTPs.

Modular, Scalable, and Customizable LC-HRMS for Exposomics.

In this chapter, we describe a multi-purpose, reversed-phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS) workflow for acquiring high-quality, non-targeted exposomics data utilizing data-dependent acquisition (DDA) combined with the use of toxicant inclusion lists for semi-targeted analysis. In addition, we describe expected retention times for >160 highly diverse xenobiotics in human plasma and serum samples. The method described is intended to serve as a generic LC-HRMS exposomics workflow for research and educational purposes. Moreover, it may be employed as a primer, allowing for further adaptations according to specialized research needs, e.g., by including reference and/or internal standards, by expanding to data-independent acquisition (DIA), or by modifying the list of compounds prioritized in fragmentation experiments (MS2).

(Pre)Clinical Metabolomics Analysis.

Metabolomics is the scientific field with the eager goal to comprehensively analyze the entirety of all small molecules of a biological system, i.e., the metabolome. Over the last few years, metabolomics has matured to become an analytical cornerstone of life science research across diverse fields, from fundamental biochemical applications to preclinical studies, including biomarker discovery and drug development. In this chapter, we provide an introduction to (pre)clinical metabolomics. We define key metabolomics aspects and provide the basis to thoroughly understand the relevance of this field in a biological and clinical context. We present and explain state-of-the-art analytical technologies devoted to metabolomic analysis as well as emerging technologies, discussing both strengths and weaknesses. Given the ever-increasing demand for handling complex datasets, the role of bioinformatics approaches in the context of metabolomic analysis is also illustrated.

Stable Isotope Tracing Experiments Using LC-MS.

Metabolomics has emerged as a pivotal field in understanding cellular function, particularly in the context of disease. In numerous diseases, including cancer, alterations in metabolism play an essential role in disease progression and drug response. Hence, unraveling the metabolic rewiring is of importance to find novel diagnostic and therapeutic strategies. Isotope tracing is a powerful technique for delving deeper into the metabolic wiring of cells. By tracking an isotopically labeled substrate through biochemical reactions in the cell, this technique provides a dynamic understanding of cellular metabolism. This chapter outlines a robust isotope tracing protocol utilizing high-resolution mass spectrometry coupled to liquid chromatography in cell culture-based models. We cover essential aspects of experimental design and analyses, providing a valuable resource for researchers aiming to employ isotopic tracing.

Quantification of Total Ketone Bodies in Biological Matrices Using Stable Isotope-Labeled Internal Standards and RP-UHPLC-MS/MS.

Acetoacetate (AcAc) and D-beta-hydroxybutyrate (D-ßOHB), the two major ketone bodies found in circulation, are linked to multiple physiological and pathophysiological states. Therefore, analytical methodologies surrounding the quantification of total ketone body (TKB) concentrations in biological matrices are paramount. Traditional methods to quantify TKBs relied on indirect spectrophotometric assays with narrow dynamic ranges, which have been significantly improved upon by modern mass spectrometry (MS)-based approaches. However, the lack of stable isotope-labeled internal standards (ISs) for AcAc and the need to distinguish D-ßOHB from its closely related structural and enantiomeric isomers pose significant obstacles. Here, we provide a protocol to synthesize and quantify a [13C] stable isotope-labeled IS for AcAc, which, in conjunction with a commercially available [2H] stable isotope-labeled IS for ßOHB, allows TKBs to be measured across multiple biological matrices. This rapid (7 min) analysis employs reverse phase ultra-high performance liquid chromatography (RP-UHPLC) coupled to tandem MS (MS/MS) to distinguish ßOHB from three structural isomers using parallel reaction monitoring (PRM), providing excellent specificity and selectivity. Finally, a method is provided that distinguishes D-ßOHB from L-ßOHB using a simple one-step derivatization to produce the corresponding diastereomers, which can be chromatographically resolved using the same rapid RP-UHPLC separation with new PRM transitions. In summary, this method provides a rigorous analytical pipeline for the analysis of TKBs in biological matrices via leveraging two authentic stable isotope-labeled ISs and RP-UHPLC-MS/MS.

A Method for Analysis of Oxidized Phospholipids from Biological Samples Using Mass Spectrometry.

Oxidized phospholipids (oxPLs) are generated during innate immunity and inflammation, where they play a variety of biological roles, including regulation of autoimmunity and coagulation. Some are generated by enzymatic reactions, leading to stereo- and regiospecificity, while many others can be formed through nonenzymatic oxidation and truncation and can be used as biomarkers of oxidative stress. Mass spectrometry methods have been developed over many years for oxPL analysis, which can provide robust estimations of molecular species and amounts, where standards are available. Here we present a method used for the analysis of enzymatically-generated oxPL (eoxPL), which allows quantification of mono-hydroxy oxylipin-containing species. We also show profiling of many other partially characterized structures in tissue samples and provide typical chromatograms obtained.

Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic Quantitation.

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 µm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.

Long Chain Base Profiling with Multiple Reaction Monitoring Mass Spectrometry.

Sphingolipids (SLs) are essential lipids with important functions in membrane formation and cell signaling. The presence of a long chain base (LCB) structure is common to all SLs. De novo SL synthesis is initiated by the enzyme serine-palmitoyltransferase (SPT), which forms an LCB by the conjugation from serine and fatty acyl-CoAs. SPT can metabolize a variety of acyl-CoA substrates, which form diverse LCB structures within and across species. The LCB then undergoes further metabolic modifications resulting in an extraordinarily diverse spectrum of sphingolipids formed. SL analysis, using liquid chromatography-mass spectrometry (LC-MS)-based methods, poses challenges due to the diverse range of frequently isobaric species. This complexity complicates the identification of underlying LCB structures using standard lipidomics approaches. Here, we describe a simplified method to analyze the LCB profile in cells, tissue, and blood. The procedure involves chemical hydrolysis to remove the conjugated headgroups and N-acyl chains, allowing to specifically resolve the underlying LCB structures by LC-MS. This method can also be combined with an isotope labeling approach to determine in vivo SPT activity and total SL de novo synthesis over time.

Sphingolipid Analysis in Clinical Research.

Sphingolipids are the most diverse class of lipids due to the numerous variations in their structural components. This diversity is also reflected in their extremely different functions. Sphingolipids are not only constituents of cell membranes but have emerged as key signaling molecules involved in a variety of cellular functions, such as cell growth and differentiation, proliferation and apoptotic cell death. Lipidomic analyses in clinical research have identified pathways and products of sphingolipid metabolism that are altered in several human pathologies. In this article, we describe how to properly design a lipidomic experiment in clinical research, how to handle plasma and serum samples for this purpose, and how to measure sphingolipids using liquid chromatography-mass spectrometry.

Nonenzymatic Oxidized Polyunsaturated Fatty Acid Products in Human Plasma and Urine Samples by LC-QTOF-MS/MS.

Oxidative stress induces autooxidation of polyunsaturated fatty acids, producing numerous isoprostanoids and isofuranoids. These oxidized products are measurable in human plasma and urine and serve as oxidative stress biomarkers for chronic diseases. This chapter details the preparation and measurement of α-linolenic acid-derived phytoprostanes and phytofurans in human samples using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS/MS).

Quantification of Octadecanoids in Human Plasma Using Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry.

Octadecanoids are a subset of oxylipins derived from 18-carbon fatty acids. These compounds have historically been understudied but have more recently attracted attention to their purported biological activity. One obstacle to the study of octadecanoids has been a lack of specific analytical methods for their measurement. A particular limitation has been the need for chiral-based methods that enable separation and quantification of individual stereoisomers. The use of chirality provides an additional dimension for distinguishing analytes produced enzymatically from those formed through autoxidation. In this chapter, we describe a comprehensive method using chiral supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) for the quantification of octadecanoids in human plasma. This method stands as an effective approach for quantifying octadecanoids and is applicable to diverse research applications including clinical research.

Recommendations for Accurate Lipid Annotation and Semi-absolute Quantification from LC-MS/MS Datasets.

Recent developments in LC-MS instrumentation and analytical technologies together with bioinformatics tools supporting high-throughput processing of large omics datasets significantly enhanced our capabilities and efficiency of identification and quantification of lipids in diverse biological materials. However, each biological matrix is characterized by its unique lipid composition, thus requiring optimization of analytical and bioinformatics workflows for each studied lipidome. Here, we describe an integrated workflow for deep lipidome profiling, accurate annotation, and semi-absolute quantification of complex lipidomes based on reversed phase chromatography and high resolution mass spectrometry. This chapter provides details on selection of the optimal extraction protocol, acquisition of LC-MS/MS data for accurate annotation of lipid molecular species, and design of lipidome-specific mixtures of internal standards to assist quantitative analysis of complex, native lipidomes.

Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100 × 3 mm; 1.7 µm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4 min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8 min including column equilibration, which enables the analysis of 160 samples per day.