Analysis Of Small Molecules Research Articles

Rongrong Pan.

"A turning point in my career was the development of nanokits for the analysis of small molecules and protein activity in single cells … I'm always in a good mood when I have a hearty meal and chat with my family after a busy day." Find out more about Rongrong Pan in her Introducing … Profile.

Simultaneous analysis of highly polar and multi-residue-type pesticides by heart-cutting 2D-LC-MS

Liquid chromatography-mass spectrometry (LC-MS) is currently the gold-standard technique for the analysis of non-volatile small organic molecules. However, one-dimensional liquid chromatography (1D-LC) cannot efficiently deal with mixtures of analytes with different physicochemical properties and, thus, specific chromatographic behaviour. As an alternative, this work proposes a two-dimensional liquid chromatography/high-resolution mass spectrometry (2D-LC-HRMS) approach for the simultaneous analysis of compounds with different polarities. It is based on the combination of hydrophilic interaction liquid chromatography (HILIC) in the first dimension (1D) and reversed-phase chromatography (RPLC) in the second dimension (2D), employing the heart-cutting methodology. The coupling between 1D and 2D was performed by a multiple heart-cutting (MHC) interface equipped with an active solvent modulation (ASM) valve. The aim of the study was the development of a 2D-LC methodology able to (i) acquire the 1D and 2D content by MS in a single analytical run, avoiding the loss of information caused by the MHC algorithm for filling the sampling loops; (ii) overcome the breakthrough problem caused by solvent incompatibility, modifying the 2D gradient during the ASM phase for this purpose. To evaluate the 2D-LC approach, pesticide residue analysis was proposed, selecting 20 pesticides covering a wide range of polarities (log Kow from −3.2 to 4.3) and including some of the so-called single residue method pesticides because of the difficulty of including them in 1D-LC multi-residue methods with satisfactory chromatographic resolution. The proposed strategy was to transfer in a single cut the void volume from the HILIC separation (consisting of the nonpolar pesticides) to the 2D for analysis under RPLC conditions. The developed assembly was assessed in a vegetable matrix (tomato) employing a hybrid QuEChERS/QuPPe sample treatment based on acetonitrile and methanol extraction. The proposed setup may be extended for 2D-LC applications where it is essential to acquire the entire content of both dimensions in a single data file just by coupling a selection valve to the MHC interface.

Recent advances in understanding brain cancer metabolomics: a review.

Regardless of the significant progress made in surgical techniques and adjuvant therapies, brain tumors are a major contributor to cancer-related morbidity and mortality in both pediatric and adult populations. Gliomas represent a significant proportion of cerebral neoplasms, exhibiting diverse levels of malignancy. The etiology and mechanisms of resistance of this malignancy are inadequately comprehended, and the optimization of patient diagnosis and prognosis is a challenge due to the diversity of the disease and the restricted availability of therapeutic options. Metabolomics refers to the comprehensive analysis of endogenous and exogenous small molecules, both in a targeted and untargeted manner, that enables the characterization of an individual's phenotype and offers valuable insights into cellular activity, particularly in the context of cancer biology, including brain tumor biology. Metabolomics has garnered attention in current years due to its potential to facilitate comprehension of the dynamic spatiotemporal regulatory network of enzymes and metabolites that enables cancer cells to adapt to their environment and foster the development of tumors. Metabolic changes are widely acknowledged as a significant characteristic for tracking the advancement of diseases, treatment efficacy, and identifying novel molecular targets for successful medical management. Metabolomics has emerged as an exciting area for personalized medicine and drug discovery, utilizing advanced analytical techniques such as nuclear magnetic resonance spectroscopy (MRS) and mass spectrometry (MS) to achieve high-throughput analysis. This review examines and highlights the latest developments in MRS, MS, and other technologies in studying human brain tumor metabolomics.

In Silico Structural-based Virtual Screening and Molecular Docking Analysis of Plant-derived Small Molecules for Treatment of COVID-19

Background and Objectives Despite many studies on the new Coronavirus disease 2019 (COVID-19), there is still a rate of disease mortality, which has made researchers to focus more on finding successful antiviral drugs. In this regard, small molecule inhibitors have been suggested for their higher safety, lower toxicity, and cost-effectiveness. In this study, we applied virtual screening and docking analysis to identify the prospective inhibitors of 3CL protease and ACE-2 receptors Subjects and Methods In this study, 1,600 natural compounds were assessed by virtual screening. The ligands with a high affinity to bind to active site residues of target proteins were identified using the glide docking method and then were included in the induced-fit docking analysis in Schrödinger-Maestro software. Results The found compounds such as Theaflavin and Delphinidin had a high affinity to bind to the receptors. They had higher binding energy and a potent inhibitory effect compared to common drugs such as chloroquine. Conclusion The introduced natural compounds can be used to suppress COVID-19. The results may help develop new drugs or formulations to combat COVID-19; however, clinical trials are needed to examine the potential of these small molecules alone or in combination with other medical procedures.

A Metasurface Plasmonic Analysis Platform Combined with Gold Nanoparticles for Ultrasensitive Quantitative Detection of Small Molecules.

Food safety related to drug residues in food has become a widespread public concern. Small-molecule drug residue analysis often relies on mass spectrometry, thin-layer chromatography, or enzyme-linked immunosorbent assays (ELISA). Some of these techniques have limited sensitivity and accuracy, while others are time-consuming, costly, and rely on specialized equipment that requires skilled operation. Therefore, the development of a sensitive, fast, and easy-to-operate biosensor could provide an accessible alternative to conventional small-molecule analysis. Here, we developed a nanocup array-enhanced metasurface plasmon resonance (MetaSPR) chip coupled with gold nanoparticles (AuNPs) (MSPRAN) to detect small molecules. As sulfamethazine drug residues in poultry eggs may cause health issues, we selected this as a model to evaluate the feasibility of using MSPRAN for small-molecule detection. The MSPRAN biosensor employed competitive immunoassay technology for sulfamethazine detection. The limit of detection was calculated as 73 pg/mL, with sensitivity approximately twice that of previously reported detection methods. Additionally, the recovery rate of the biosensor, tested in egg samples, was similar to that measured using ELISA. Overall, this newly developed MSPRAN biosensor platform for small-molecule detection provides fast and reliable results, facile operation, and is relatively cost-effective for application in food safety testing, environmental monitoring, or clinical diagnostics.

P-686 Long photoperiod exposure results in glycometabolism disorder in the cumulus-oocyte complex in SD rats

Abstract Study question Does long photoperiod exposure cause glycometabolism changes in the cumulus-oocyte complex? Summary answer The levels of key enzymes and substrates involved in glycometabolism have changed in the cumulus-oocyte complex in SD rats due to circadian rhythm changes. What is known already Circadian rhythm is essential for maintaining the required reproductive activities in mammalians. In our previous research, long photoperiod exposure results in the reduction of ovulation number and the increase of ROS level in oocytes. In the process of oocyte maturation, the tricarboxylic acid cycle and energy metabolism gradually increase, while the ability of the oocyte to absorb glucose is low. It is mainly metabolized by cumulus granulosa cells into pyruvate and then transported to oocytes for subsequent metabolism. Metabolomics is a comprehensive analysis of small molecules, which provides key information about cellular metabolic states. Study design, size, duration Female SD rats, 6-8 weeks old, were randomized into two groups. The rats (n = 20) were exposed to the control photoperiod (12 hours light/12 hours dark) or long photoperiod (16 hours light/8 hours dark) for 12 weeks. Participants/materials, setting, methods Cumulus-oocyte complexes were collected after ovarian stimulation and numbers were counted. The parameters compared include MII rate, fertilization rate, and ovarian follicle number. We used mass spectrometry-based metabolomics to directly measure metabolite abundance. The glycometabolism pathway and its regulatory signaling molecules were analyzed by qPCR and/or Western blots. Main results and the role of chance Long photoperiod exposure significantly reduced the ovulation rate, MII rate and fertilization rate. Interestingly, we find secondary follicle and antral follicle were decreased (p < 0.01), while primordial follicle and primary follicle were increased (p < 0.01). We identified a total of 318 metabolites in COC. Among them, 59 metabolite levels have significant differences between the two groups (FDR < 0.05). The metabolical function analysis suggested that the phosphatidylinositol phosphate metabolism, pyruvate metabolism, phosphatidylcholine biosynthesis, and glycolysis pathways were influenced by circadian interference. In addition, hexokinase, glucose-6-phosphate isomerase, phosphofructokinase glyceraldehyde 3-phosphate expression level were down regulated. Limitations, reasons for caution More studies are required to address how clock gene regulating glycometabolism process. And Translating results in rat model to the human setting is a limitation of this study. Wider implications of the findings Our study findings are first to demonstrate that glycolmetabolism is regulated by long photoperiod exposure in follicle development. This knowledge could help to develop strategies to modulate specific recovery strategies based on metabolic regulator to improve human IVF. Trial registration number Not applicable

Step-by-Step Approach to Build Multiple Reaction Monitoring (MRM) Profiling Instrument Acquisition Methods for Class-based Lipid Exploratory Analysis by Mass Spectrometry.

Multiple reaction monitoring (MRM) profiling is a strategy for the exploratory analysis of small molecules and lipids by direct sample injection, ie, without the use of chromatographic separation. It is based on instrument methods that comprise a list of ion transitions (MRMs), in which the precursor ion is the expected ionized m/z of the lipid at its species level, ie, the description of lipid class and number of carbon and double bonds in the fatty acid chain(s), and the product ion is a fragment expected for the lipid class or for the fatty acid neutral loss. The Lipid Maps database is expanding constantly, and therefore the MRM-profiling methods associated with this database need to be continuously updated. Here, we provide a comprehensive overview and the key references for the MRM-profiling methodology and workflow, followed by a step-by-step approach to build MRM-profiling instrument acquisition methods for class-based lipid exploratory analysis based on the Lipid Maps database. The detailed workflow includes (1) importing the list of lipids from the database; (2) for a given class, combining isomeric lipids described at full structural level into 1 entry to obtain the neutral mass at species level; (3) attributing the standard Lipid Maps abbreviated nomenclature for the lipid at its species level; (4) predicting the ionized precursor ions; and (5) adding the expected product ion. We also describe how to simulate the precursor ion for the suspect screening of modified lipids using lipid oxidation and their expected product ions as an example. After determining the MRMs, information about collision energy, dwell time, and other instrument parameters are added to finalize the acquisition method. As an example of final method output, we describe the format for Agilent MassHunter v.B.06 and provide the parameters in which optimization can be performed by lipid class using one or more lipid standards.

Poly(L-dopa) film-assisted laser desorption/ionization of small biomolecules and drugs

Although matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) owns unique merits, the applicability of MALDI for small molecule analysis is still very limited due to the matrix-related interferences in low-mass region. We present a new strategy for the analysis of small biomolecules and drugs by LDI MS. Poly(L-dopa) film was in situ polymerized on standard stainless steel MALDI plate, and the analytes deposited on the film were vaporized and ionized under laser irradiation. Poly(L-dopa) film was found highly effective for laser desorption/ionization of small molecules including amino acids, organic acids, nucleosides, monosaccharides and sulfa drugs at pmol levels with extremely low background noise. Amino acids was detectable even in saturated salt solution. The good salt tolerance and high sensitivity allowed its application to the detection of celecoxib in serum at concentration as low as 0.29 μM. The preparation of poly(L-dopa) film is very simple and convenient. It is believed that poly(L-dopa) film-assisted laser desorption/ionization mass spectrometry has potential to be a practical solution for small biomolecule analysis.

Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry.

Dried blood spot samples are simple to prepare and transport, enabling safe and accessible diagnostics, both locally and globally. We review dried blood spot samples for clinical analysis, focusing on liquid chromatography-mass spectrometry as a versatile measurement tool for these samples. Dried blood spot samples can provide information for, for example, metabolomics, xenobiotic analysis, and proteomics. Targeted analyses of small molecules are the main application of dried blood spot samples and liquid chromatography-mass spectrometry, but emerging applications include untargeted metabolomics and proteomics. Applications are highly varied, including analyses related to newborn screening, diagnostics and monitoring of disease progression and treatment effects of virtually any disease, as well as studies into the physiology and effects of diet, exercise, xenobiotics, and doping. A range of dried blood spot products and methods are available, and applied liquid chromatography-mass spectrometry instrumentation is varied with regard to liquid chromatography column formats and selectivity. In addition, novel approaches such as on-paper sample preparation (e.g., selective trapping of analytes with paper-immobilized antibodies) are described. We focus on research papers published in the last 5 years.

Metal-Organic Framework-Decorated Nanochannel Electrode: Integration of Internal Nanoconfined Space and Outer Surface for Small-Molecule Sensing.

Ionic current measurement has been the dominant signaling strategy in nanochannel-based sensors. However, the direct probing of the capture of small molecules is still challenging, and the sensing potential of the outer surface of nanochannels is always ignored. Here, we report the fabrication of an integrated nanochannel electrode (INCE) with nanoporous gold layers modified on two sides of nanochannels, and its application for small-molecule analysis was explored. Metal-organic frameworks (MOFs) were decorated inside and outside of nanochannels, enabling the reduction of pore size to several nanometers, which is among the thickness range of the electric double layer for confined ion diffusion. Combined with excellent adsorption characteristics of MOFs, the developed nanochannel sensor successfully constructed the internal nanoconfined space that could directly capture small molecules and instantly generate a current signal. The contribution of the outer surface and the internal nanoconfined space to diffusion suppression to electrochemical probes was investigated. We found that the constructed nanoelectrochemical cell was sensitive in both the inner channel and the outer surface, signifying a novel sensing mode with integration of the internal nanoconfined space and the outer surface of nanochannels. The MOF/INCE sensor showed excellent performance toward tetracycline (TC) with a detection limit of 0.1 ng·mL-1. Subsequently, sensitive and quantitative detection of TC down to 0.5 μg·kg-1 was achieved in actual chicken samples. This work may open up a new model of nanoelectrochemistry and provide an alternative solution in the field of nanopore analysis for small molecules.

Reversed HILIC Gradient: A Powerful Strategy for On-Line Comprehensive 2D-LC.

The aim of the present work is to evaluate the possibilities and limitations of reversed hydrophilic interaction chromatography (revHILIC) mode in liquid chromatography (LC). This chromatographic mode consists of combining a highly polar stationary phase (bare silica) with a gradient varying from very low (1-5%) to high (40%) acetonitrile content (reversed gradient compared to HILIC). The retention behavior of revHILIC was first compared with that of reversed-phase LC (RPLC) and HILIC using representative mixtures of peptides and pharmaceutical compounds. It appears that the achievable selectivity can be ranked in the order RPLC > revHILIC > HILIC with the two different samples. Next, two-dimensional liquid chromatography (2D-LC) conditions were evaluated by combining RPLC, revHILIC, or HILIC with RPLC in an on-line comprehensive (LC × LC) mode. evHILIC × RPLC not only showed impressive performance in terms of peak capacity and sensitivity, but also provided complementary selectivity compared to RPLC × RPLC and HILIC × RPLC. Indeed, both the elution order and the retention time range differ significantly between the three techniques. In conclusion, there is no doubt that revHILIC should be considered as a viable option for 2D-LC analysis of small molecules and also peptides.

Submicron Nonporous Silica Particles for Enhanced Separation Performance in pCEC.

Applications of submicron-scale particles are of rising interest in separation science due to their favorable surface-to-volume ratio and their fabrication of highly ordered structures. The uniformly dense packing beds in columns assembled from nanoparticles combined with an electroosmotic flow-driven system has great potential in a highly efficient separation system. Here, we packed capillary columns using a gravity method with synthesized nanoscale C18-SiO2 particles having diameters of 300-900 nm. The separation of small molecules and proteins was evaluated in the packed columns on a pressurized capillary electrochromatography platform. The run-to-run reproducibility regarding retention time and peak area for the PAHs using a column packed with 300 nm C18-SiO2 particles were less than 1.61% and 3.17%, respectively. Our study exhibited a systematic separation analysis of small molecules and proteins based on the columns packed with submicron particles combined with the pressurized capillary electrochromatography (pCEC) platform. This study may provide a promising analytical approach with extraordinary column efficiency, resolution, and speed for the separation of complex samples.

An efficient Pt@MXene platform for the analysis of small-molecule natural products

Small-molecule (m/z<500) natural products have rich biological activity and significant application value thus need to be effectively detected. Surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) has become a powerful detection tool for small-molecule analysis. However, more efficient substrates need to be developed to improve the efficiency of SALDI MS. Thus, platinum nanoparticle-decorated Ti3C2 MXene (Pt@MXene) was synthesized in this study as an ideal substrate for SALDI MS in positive ion mode and exhibited excellent performance for the high-throughput detection of small molecules. Compared with using MXene, GO, and CHCA matrix, a stronger signal peak intensity and wider molecular coverage was obtained using Pt@MXene in the detection of small-molecule natural products, with a lower background, excellent salt and protein tolerance, good repeatability, and high detection sensitivity. The Pt@MXene substrate was also successfully used to quantify target molecules in medicinal plants. The proposed method has potentially wide application.

Detection and analysis of chiral molecules as disease biomarkers.

The chirality of small metabolic molecules is important in controlling physiological processes and indicating the health status of humans. Abnormal enantiomeric ratios of chiral molecules in biofluids and tissues occur in many diseases, including cancers and kidney and brain diseases. Thus, chiral small molecules are promising biomarkers for disease diagnosis, prognosis, adverse drug-effect monitoring, pharmacodynamic studies and personalized medicine. However, it remains difficult to achieve cost-effective and reliable analysis of small chiral molecules in clinical procedures, in part owing to their large variety and low concentration. In this Review, we describe current and emerging techniques that detect and quantify small-molecule enantiomers and their biological importance.

Au@BN-enhanced laser desorption/ionization mass spectrometry and imaging for determination of fipronil and its metabolites in food and biological samples

Gold nanoparticles (AuNPs) represent an attractive inorganic matrix for laser desorption/ionization mass spectrometry (LDI-MS) detection of low-molecular-weight analytes; however, their direct use is hindered by severe aggregation. To limit AuNPs aggregation, hexagonal boron nitride nanosheets (h-BNNs) were employed as supports to improve their desorption/ionization efficiency. Thus, Au@BN was synthesized and systematically characterized. It showed low background noise and high sensitivity for LDI-MS of fipronil and its metabolites. Au@BN-assisted LDI-MS was validated using complex samples including blueberry juice, green tea beverage, and fish muscle, achieving low detection limits (0.05–0.20 µg·L−1 for liquid media, 0.82–1.25 ng·g−1 for fish muscle), wide linear ranges (0.2–100 µg·L−1 for liquid media, 3.00–1000 ng·g−1 for fish muscle), high reproducibility (7.55%–13.7%), and satisfactory recoveries (82.62%–109.1%). Furthermore, spatial distributions of analytes in strawberries and zebrafish were successfully imaged. This strategy allows for the quantitative analysis of other small molecules in complex substrates.

Holistic quality evaluation of Callicarpae Formosanae Folium by multi-chromatography-based qualitative and quantitative analysis of polysaccharides and small molecules

Callicarpae Formosanae Folium (CFF), derived from the leaves of Callicarpa formosana Rolfe, is a common Chinese medicinal herb used for the treatment of hematemesis. Phytochemical studies found that phenylpropanoids, flavonoids, terpenoids and polysaccharides were the main ingredients of CFF. However, there is limited scientific information concerning holistic quality method and quality consistency evaluation of CFF. In this study, a strategy integrating HPGPC-ELSD, HPLC-PDA, UV–VIS and UPLC-QTOF-MS/MS was firstly developed to simultaneously qualify and quantify polysaccharides, as well as representative small molecules in CFF. HPGPC-ELSD was applied to characterize the molecular weight distribution of polysaccharides, HPLC-PDA was developed to qualitatively and quantitatively determine monosaccharides. UV-VIS was used to determine the total polysaccharides content, and UPLC-QTOF-MS/MS was established to characterize the small molecules. The quality consistency of commercial CFF (CM-CFF) was also evaluated. It was shown that the relative molecular weights, the compositional monosaccharides and small molecules composition in CM-CFF and self-collected CFF (SC-CFF) samples were similar. A total of 32 small molecules including 6 phenylpropanoids, 7 flavonoids and 19 terpenoids were characterized in CFF. However, the variation was observed in the content of polysaccharides, luteolin, ursolic acid, as well as total contents of terponoids in CM-CFF samples, which implied that the holistic quality of CM-CFF was inconsistent. The results suggested that the proposed evaluation strategy could be applied as a potential approach for the quality control of CFF. And the quality of CM-CFF should be improved by Good Agriculture Practice (GAP) base and standard processing method.

Label-free biosensing of lignans for therapeutics using engineered model surfaces

The label-free interaction analysis of macromolecules and small molecules has increasing importance nowadays, both in diagnostics and therapeutics. In the blood vascular system, human serum albumin (HSA) is a vital globular transport protein with potential multiple ligand binding sites. Characterizing the binding affinity of compounds to HSA is essential in pharmaceutics and in developing new compounds for clinical application. Aryltetralin lignans from the roots of Anthriscus sylvestris are potential antitumor therapeutic candidates, but their molecular scale interactions with specific biomolecules are unrevealed. Here, we applied the label-free grating-coupled interferometry (GCI) biosensing method with a polycarboxylate-based hydrogel layer with immobilized HSA on top of it. With this engineered model surface, we could determine the binding parameters of two novel aryltetralin lignans, deoxypodophyllotoxin (DPT), and angeloyl podophyllotoxin (APT) to HSA. Exploiting the multi-channel referencing ability, the unique surface sensitivity, and the throughput of GCI, we first revealed the specific biomolecular interactions. Traditional label-free kinetic measurements were also compared with a novel, fast way of measuring affinity kinetics using less sample material (repeated analyte pulses of increasing duration (RAPID)). Experiments with well-characterized molecular interactions (furosemide to carbonic-anhydrase (CAII) and warfarin, norfloxacin to HSA) were performed to prove the reliability of the RAPID method. In all investigated cases, the RAPID and traditional measurement gave similar affinity values. In the case of DPT, the measurements and relevant modeling suggested two binding sites on HSA, with dissociation constant values of Kd1 = 1.8 ± 0.01 μM, Kd2 = 3 ± 0.02 μM. In the case of APT, the experiments resulted in Kd1 = 9 ± 1.7 μM, Kd2 = 28 ± 0.3 μM. The obtained binding values might suggest the potential medical application of DPT and APT without further optimization of their binding affinity to HSA. These results could be also adapted to other biomolecules and applications where sample consumption and the rapidity of the measurements are critical.

The secret of reversed-phase/weak cation exchange retention mechanisms in mixed-mode liquid chromatography applied for small drug molecule analysis

Resolving complex sample mixtures by liquid chromatography in a single run is challenging. The so-called mixed-mode liquid chromatography (MMLC) which combines several retention mechanisms within a single column, can provide resource-efficient separation of solutes of diverse nature. The Acclaim Mixed-Mode WCX-1 column, encompassing hydrophobic and weak cation exchange interactions, was employed for the analysis of small drug molecules. The stationary phase's interaction abilities were assessed by analysing molecules of different ionisation potentials. Mixed Quantitative Structure-Retention Relationship (QSRR) models were developed for revealing significant experimental parameters (EPs) and molecular features governing molecular retention. According to the plan of Face-Centred Central Composite Design, EPs (column temperature, acetonitrile content, pH and buffer concentration of aqueous mobile phase) variations were included in QSRR modelling. QSRRs were developed upon the whole data set (global model) and upon discrete parts, related to similarly ionized analytes (local models) by applying gradient boosted trees as a regression tool. Root mean squared errors of prediction for global and local QSRR models for cations, anions and neutrals were respectively 0.131; 0.105; 0.102 and 0.042 with the coefficient of determination 0.947; 0.872; 0.954 and 0.996, indicating satisfactory performances of all models, with slightly better accuracy of local ones. The research showed that influences of EPs were dependant on the molecule's ionisation potential. The molecular descriptors highlighted by models pointed out that electrostatic and hydrophobic interactions and hydrogen bonds participate in the retention process. The molecule's conformation significance was evaluated along with the topological relationship between the interaction centres, explicitly determined for each molecular species through local models. All models showed good molecular retention predictability thus showing potential for facilitating the method development.

Perspectives and opinions from scientific leaders on the evolution of data-independent acquisition for quantitative proteomics and novel biological applications

The methodology of data-independent acquisition (DIA) within mass spectrometry (MS) was developed into a method of choice for quantitative proteomics, to capture the depth and dynamics of biological systems, and to perform large-scale protein quantification. DIA provides deep quantitative proteome coverage with high sensitivity, high quantitative accuracy, and excellent acquisition-to-acquisition reproducibility. DIA workflows benefited from the latest advancements in MS instrumentation, acquisition/isolation schemes, and computational algorithms, which have further improved data quality and sample throughput. This powerful DIA-MS scan type selects all precursor ions contained in pre-determined isolation windows, and systematically fragments all precursor ions from each window by tandem mass spectrometry, subsequently covering the entire precursor ion m/z range. Comprehensive proteolytic peptide identification and label-free quantification are achieved post-acquisition using spectral library-based or library-free approaches. To celebrate the &gt; 10 years of success of this quantitative DIA workflow, we interviewed some of the scientific leaders who have provided crucial improvements to DIA, to the quantification accuracy and proteome depth achieved, and who have explored DIA applications across a wide range of biology. We discuss acquisition strategies that improve specificity using different isolation schemes, and that reduce complexity by combining DIA with sophisticated chromatography or ion mobility separation. Significant leaps forward were achieved by evolving data processing strategies, such as library-free processing, and machine learning to interrogate data more deeply. Finally, we highlight some of the diverse biological applications that use DIA-MS methods, including large-scale quantitative proteomics, post-translational modification studies, single-cell analysis, food science, forensics, and small molecule analysis.

Environmental proteomics: a potential tool in wastewater-based epidemiology

Wastewater-based epidemiology (WBE) is a new complementary alternative to traditional epidemiological surveillance approaches for community health surveys. It provides objective, flexible, and comprehensive data on diseases, exposure to toxicants and pathogens, social habits, and lifestyle. Wastewater is regarded as an integrated pooled sample of the entire population served by a certain wastewater system, thus providing an average picture of its health status. WBE relies on the extraction, analysis, and subsequent interpretation of appropriate health status indicators, including both chemical and biological components, either exogenous or endogenous, altogether constituting the exposome. Instrumental development, especially mass spectrometry (MS) for the chemical analysis of small and large molecules, has been critical to the success achieved through WBE. Whereas all previous studies have been restricted to small molecules, the use of proteomics in WBE is an obvious extension of the intensive ongoing pursuit of biomarkers for community surveillance of diseases and health status. The relevance of human proteins present in wastewater as potential WBE indicators of population-prevalent diseases has been previously postulated, although no real examples to demonstrate their feasibility have been available until recently. These findings provide new insights into the characterization of wastewater proteomics useful for practical purposes related not only to WBE applications but also to environmental monitoring and wastewater treatment plant management.