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Thanks to the plummeting costs of continuously evolving omics analytical platforms, research centers collect multiomics data more routinely. They are, however, confronted with the lack of a versatile software solution to harmoniously analyze single-omics and interpret multiomics data. We have developed iSODA, a web-based application for the analysis of single- and multiomics data. The tool emphasizes intuitive interactive visualizations designed for user-driven data exploration. Researchers can access a variety of functions ranging from simple visualization like volcano plots and PCA to advanced functional analyses like enrichment analysis and lipid saturation analysis. For integrated multiomics, iSODA incorporates multi-omics factor analysis and similarity network fusion. The ability to adapt the data on-the-fly allows for tasks, such as the removal of outlier samples or failed features, imputation, or normalization. All results are presented through interactive plots, the modular design supports extensions, and tooltips and tutorials provide comprehensive guidance for users. iSODA is accessible under http://isoda.online/.

PubChem serves as a comprehensive repository, housing over 100 million unique chemical structures representing the breadth of our chemical knowledge across numerous fields including metabolism, pharmaceuticals, toxicology, cosmetics, agriculture, and many more. Rapid identification of these small molecules increasingly relies on electrospray ionization (ESI) paired with tandem mass spectrometry (MS/MS), particularly by comparison to genuine standard MS/MS data sets. Despite its widespread application, achieving consistency in MS/MS data across various analytical platforms remains an unaddressed concern. This study evaluated MS/MS data derived from one hundred molecular standards utilizing instruments from five manufacturers, inclusive of quadrupole time-of-flight (QTOF) and quadrupole orbitrap "exactive" (QE) mass spectrometers by Agilent (QTOF), Bruker (QTOF), SCIEX (QTOF), Waters (QTOF), and Thermo QE. We assessed fragment ion variations at multiple collisional energies (0, 10, 20, and 40 eV) using the cosine scoring algorithm for comparisons and the number of fragments observed. A parallel visual analysis of the MS/MS spectra across instruments was conducted, consistent with a standard procedure that is used to circumvent the still prevalent issue of mischaracterizations as shown for dimethyl sphingosine and C20 sphingosine. Our analysis revealed a notable consistency in MS/MS data and identifications, with fragment ions' m / z values exhibiting the highest concordance between instrument platforms at 20 eV, the other collisional energies (0, 10, and 40 eV) were significantly lower. While moving toward a standardized ESI MS/MS protocol is required for dependable molecular characterization, our results also underscore the continued importance of corroborating MS/MS data against standards to ensure accurate identifications. Our findings suggest that ESI MS/MS manufacturers, akin to the established norms for gas chromatography mass spectrometry instruments, should standardize the collision energy at 20 eV across different instrument platforms.

Lipid analysis at the molecular species level represents a valuable opportunity for clinical applications due to the essential roles that lipids play in metabolic health. However, a comprehensive and high-throughput lipid profiling remains challenging given the lipid structural complexity and exceptional diversity. Herein, we present an 'omic-scale targeted LC-MS/MS approach for the straightforward and high-throughput quantification of a broad panel of complex lipid species across 26 lipid (sub)classes. The workflow involves an automated single-step extraction with 2-propanol, followed by lipid analysis using hydrophilic interaction liquid chromatography in a dual-column setup coupled to tandem mass spectrometry with data acquisition in the timed-selective reaction monitoring mode (12 min total run time). The analysis pipeline consists of an initial screen of 1903 lipid species, followed by high-throughput quantification of robustly detected species. Lipid quantification is achieved by a single-point calibration with 75 isotopically labeled standards representative of different lipid classes, covering lipid species with diverse acyl/alkyl chain lengths and unsaturation degrees. When applied to human plasma, 795 lipid species were measured with median intra- and inter-day precisions of 8.5 and 10.9%, respectively, evaluated within a single and across multiple batches. The concentration ranges measured in NIST plasma were in accordance with the consensus intervals determined in previous ring-trials. Finally, to benchmark our workflow, we characterized NIST plasma materials with different clinical and ethnic backgrounds and analyzed a sub-set of sera ( n = 81) from a clinically healthy elderly population. Our quantitative lipidomic platform allowed for a clear distinction between different NIST materials and revealed the sex-specificity of the serum lipidome, highlighting numerous statistically significant sex differences.

The fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3) is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of DHA are poorly understood. Here, we report the design, synthesis, and application of a complementary pair of bio-orthogonal, photoreactive probes based on the polyunsaturated scaffold DHA and its oxidative metabolite 17-hydroxydocosahexaenoic acid (17-HDHA). In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific targets of the omega-3 signaling lipids in primary human macrophages. Prostaglandin reductase 1 (PTGR1) was identified as an interaction partner that metabolizes 17-oxo-DHA, an oxidative metabolite of 17-HDHA. 17-oxo-DHA reduced the formation of pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils. Our results demonstrate the potential of comparative photoaffinity protein profiling for the discovery of metabolic enzymes of bioactive lipids and highlight the power of chemical proteomics to uncover new biological insights.

Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics' technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950-Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231-Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.

Single quadrupole mass spectrometry (MS) with enhanced in-source multiple fragment ion monitoring was designed to perform high sensitivity quantitative mass analyses. Enhanced in-source fragmentation amplifies fragmentation from traditional soft electrospray ionization producing fragment ions that have been found to be identical to those generated in tandem MS. We have combined enhanced in-source fragmentation data with criteria established by the European Union Commission Directive 2002/657/EC for electron ionization single quadrupole quantitative analysis to perform quantitative analyses. These experiments were performed on multiple types of complex samples that included a mixture of 50 standards, as well as cell and plasma extracts. The dynamic range for these quantitative analyses was comparable to triple quadrupole multiple reaction monitoring (MRM) analyses at up to 5 orders of magnitude with the cell and plasma extracts showing similar matrix effects across both platforms. Amino acid and fatty acid measurements performed from certified NIST 1950 plasma with isotopically labeled standards demonstrated accuracy in the range of 91-110% for the amino acids, 76-129% for the fatty acids, and good precision (coefficient of variation <10%). To enhance specificity, a newly developed correlated ion monitoring algorithm was designed to facilitate these analyses. This algorithm autonomously processes, aligns, filters, and compiles multiple ions within one chromatogram enabling both precursor and in-source fragment ions to be correlated within a single chromatogram, also enabling the detection of coeluting species based on precursor and fragment ion ratios. Single quadrupole instrumentation can provide MRM level quantitative performance by monitoring/correlating precursor and fragment ions facilitating high sensitivity analysis on existing single quadrupole instrumentation that are generally inexpensive, easy to operate, and technically less complex.

Differential mobility spectrometry (DMS) is capable of separating stereoisomeric molecular ions based on their mobility in an oscillating electrical field with an asymmetric waveform. Thus, it is an "orthogonal" technique to chromatography and (tandem) mass spectrometry. Bioactive lipids, particularly of the eicosanoid and docosanoid class feature numerous stereoisomers, which exhibit a highly specific structure-activity relationship. Moreover, the geometry of these compounds also reflects their biochemical origin. Therefore, the unambiguous characterization of related isomers of the eicosanoid and docosanoid classes is of fundamental importance to the understanding of their origin and function in many biological processes. Here we show, that SelexION DMS technology coupled to μLC-MS/MS is capable of differentiating at least five closely related leukotrienes partially coeluting and (almost) unresolvable using LC-MS/MS only. We applied the developed method to the separation of LTB4 and its coeluting isomer 5S,12S-diHETE in murine peritoneal exudate cells, showing that LTB4 is present only after zymosan A injection while its isomer 5S,12S-diHETE is produced after saline (PBS) administration. Additionally, we show that the SelexION technology can also be applied to the separation of PD1 and PDX (10S,17S-diHDHA), two isomeric protectins.

Here, we present the application of a cross-platform approach, combining rapid direct infusion high-resolution/accurate mass electrospray ionization Fourier transform ion-cyclotron mass spectrometry (ESI-FTICRMS) with in-depth data-dependent LC-MS(2) and LC-MS(3) analysis for lipid profiling. The analytical approach as well as the subsequent data handling is described. The method was applied to human synovial fluid samples from osteo- and rheumatoid arthritis patients. Multivariate statistical analysis revealed esterified oxylipids as molecular features in a subset of the patient samples. Employing LC-MS(2) and LC-MS(3) analysis of these species, we were able to clarify the hypothesized lipid structures initially based on the accurate mass measurements performed on the ESI-FTICRMS platform. LC-MS(3) analysis of intact esterified oxy-lipids and LC-MS(2) analysis of the hydrolysis products allowed for the detection of positional isomers. The approach led to the structural elucidation of hydroxylated docosapentaenoic acid-containing diacyl-phosphatidylcholine type phospholipids in human synovial fluid.

Several proteomics approaches are available that are defined by the level (protein or peptide) at which analysis takes place. The most widely applied method still is bottom-up proteomics where the protein is digested into peptides that can be efficiently analyzed with a wide range of LC-MS or MALDI-TOF-MS instruments. Sample preparation for bottom-up proteomics experiments requires several treatment steps in order to get from the protein to the peptide level and can be very laborious. The most crucial step in such approaches is the protein digestion, which is often the bottleneck in terms of time consumption. Therefore, a significant gain in throughput may be obtained by speeding up the digestion process. Current techniques allow for reduction of the digestion time from overnight (~15 h) to minutes or even seconds. This advancement also makes integration into online systems feasible, thereby reducing the number of tedious sample handling steps and the risk of sample loss. In this review, an overview is given of the currently available digestion strategies and recent developments in the acceleration of the digestion process. Additionally, tailored approaches for classes of proteins that pose specific challenges are discussed.

The aim of the here presented study was to combine high performance liquid chromatography with plate reader technology in order to overcome certain drawbacks of integrated online systems as well as offline plate reader approaches. The described method combines an "at-line" enzyme assay for the simultaneous bioactivity determination with parallel QTOF MS data acquisition for analyte identification. All biochemical reagents are added in an online mode directly to the column effluent (postcolumn addition/mixing), and the complete screening assay mixture is subsequently microfractionated into a 1536 well plate. The screening of a natural extract fortified with two well-known Protein Kinase A inhibitors and the identification of an inhibitor in a natural extract showed the applicability of the approach to detect bioactive compounds in low concentrations in a complex mixture. The described mode of operation utilizes today's plate reader technology to its full capacity and directly hyphenates it to a high resolution separation technique which has not been shown before. Furthermore, it allows coupling of a microbore HPLC with a biochemical screening assay without compromising resolution and overcomes problems associated with the 1536 well format.