Microbore-based vacuum jacketed liquid chromatography for fast, efficient and sustainable high throughput bioanalysis.

Extra-column band broadening can significantly reduce the performance of rapid ultra-high performance liquid chromatography-MS-based methods (UHPLC-MS). However, as we show here, UHPLC-MS/MS methods on short 2.1 mm i.

Challenges in Lipidomics Biomarker Identification: Avoiding the Pitfalls and Improving Reproducibility.

Identification of features with high levels of confidence in liquid chromatography-mass spectrometry (LC-MS) lipidomics research is an essential part of biomarker discovery, but existing software platforms can give inconsistent results, even from identical spectral data. This poses a clear challenge for reproducibility in biomarker identification. In this work, we illustrate the reproducibility gap for two open-access lipidomics platforms, MS DIAL and Lipostar, finding just 14.

Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study.

Amino acids (AAs) and their metabolites are important building blocks, energy sources, and signaling molecules associated with various pathological phenotypes. The quantification of AA and tryptophan (TRP) metabolites in human serum and plasma is therefore of great diagnostic interest. Therefore, robust, reproducible sample extraction and processing workflows as well as rapid, sensitive absolute quantification are required to identify candidate biomarkers and to improve screening methods.

Detection of pharmacolipidodynamic effects following the intravenous and oral administration of gefitinib to C57Bl/6JRj mice by rapid UHPLC-MS analysis of plasma.

Omics-based biomarker technologies, including metabolic profiling (metabolomics/metabonomics) and lipidomics, are making a significant impact on disease understanding, drug development, and translational research. A wide range of patho-physiological processes involve lipids and monitoring changes in lipid abundance can give valuable insights into mechanisms of drug action, off target pharmacology and toxicity. Here we report changes, detected by untargeted LC-MS, in the plasma lipid profiles of male C57Bl/6JRj mice following the PO and IV administration of the epidermal growth factor receptor (EGFR) inhibitor gefitinib.

The rapid separation and characterization of sulfates of tyrosine and its metabolites in reaction mixtures and human urine using a cyclic ion mobility device and mass spectrometry.

Ion mobility (IM) separations, especially when combined with mass spectrometry, offer the opportunity for the rapid analysis and characterization of mixtures. However, the limited resolution afforded by many IM systems means that in practice applications may be limited. Here we have employed an IM separation on a high-resolution cyclic IM device with MS/MS to separate and characterize mixtures of sulfated isomers of tyrosine and associated metabolites containing multiple sulfated isoforms present in reaction mixtures.

Increasing coverage of the urinary polar metabolome using ultra high-performance hydrophobic interaction liquid chromatography combined with linear and cyclic travelling wave ion mobility and mass spectrometry.

The use of HILIC-based separations for the analysis of polar metabolites in metabolic phenotyping studies is well established. Here, we demonstrate the increased coverage of the polar metabolome obtained by travelling wave (TW) ion mobility (IM) instruments combined with HILIC and mass spectrometry (MS) for metabotyping rat and mouse urine samples. Profiling was performed using either a linear TW IM-MS based instrument with a path length of 40 cm or an instrument with a cyclic travelling wave analyser (cIM) with a path length of 95 cm.

Development of a single mobile phase for LC-IM-MS-based discovery lipidomics and metabolic phenotyping: Application to methapyrilene hepatotoxicity in the rat.

The untargeted global profiling of endogenous metabolites and lipids has the potential to increase knowledge and understanding in many areas of biology. LC-MS/MS is a key technology for such analyses however, several different LC methodologies, using different mobile phase compositions, are required to cover the diversity in polarity and analyte structure encountered in biological samples. Most notably many lipid screening methods make use of isopropanol (IPA) as a major component of mobile phases employed for comprehensive lipidomic profiling.

Multi-omic diagnostics of prostate cancer in the presence of benign prostatic hyperplasia.

There is an unmet need for improved diagnostic testing and risk prediction for cases of prostate cancer (PCa) to improve care and reduce overtreatment of indolent disease. Here we have analysed the serum proteome and lipidome of 262 study participants by liquid chromatography-mass spectrometry, including participants diagnosed with PCa, benign prostatic hyperplasia (BPH), or otherwise healthy volunteers, with the aim of improving biomarker specificity. Although a two-class machine learning model separated PCa from controls with sensitivity of 0.

Lipidomic Analysis Reveals Differences in Species Driven Largely by Plasmalogens, Glycerophosphoinositols and Certain Sphingolipids.

There has been increasing interest in bacterial lipids in recent years due, in part, to their emerging role as molecular signalling molecules. is an important member of the mammalian gut microbiota that has been shown to produce sphingolipids (SP) that pass through the gut epithelial barrier to impact host SP metabolism and signal into host inflammation pathways. also produces a novel family of N-acyl amines (called glycine lipids) that are potent ligands of host Toll-like receptor 2 (TLR2).

A Novel Blood Proteomic Signature for Prostate Cancer.

Prostate cancer is the most common malignant tumour in men. Improved testing for diagnosis, risk prediction, and response to treatment would improve care. Here, we identified a proteomic signature of prostate cancer in peripheral blood using data-independent acquisition mass spectrometry combined with machine learning.

A chromatographic and immunoprofiling approach to optimising workflows for extraction of gluten proteins from flour.

Ingestion of gluten proteins from wheat, and related prolamin proteins from barley, rye, and oats, can cause adverse reactions in individuals with coeliac disease and IgE-mediated allergies. As there is currently no cure for these conditions, patients must practice avoidance of gluten-containing foods. In order to support patients in making safe food choices, foods making a "gluten-free" claim must contain no more than 20 mg/Kg of gluten.

High Throughput LC-MS Platform for Large Scale Screening of Bioactive Polar Lipids in Human Plasma and Serum.

Lipids play a key role in many biological processes, and their accurate measurement is critical to unraveling the biology of diseases and human health. A high throughput HILIC-based (LC-MS) method for the semiquantitative screening of over 2000 lipids, based on over 4000 MRM transitions, was devised to produce an accessible and robust lipidomic screen for phospholipids in human plasma/serum. This methodology integrates many of the advantages of global lipid analysis with those of targeted approaches.

The Fate of IgE Epitopes and Coeliac Toxic Motifs during Simulated Gastrointestinal Digestion of Pizza Base.

Understanding how food processing may modify allergen bioaccessibility and the evolution of immunologically active peptides in the gastrointestinal tract is essential if knowledge-based approaches to reducing the allergenicity of food are to be realised. A soy-enriched wheat-based pizza base was subjected to in vitro oral-gastro-duodenal digestion and resulting digests analysed using a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The digestion profile of pizza base resembled that of bread crust where higher temperatures during baking reduced protein solubility but still resulted in the generation of a complex mixture of peptides.

Access to the Phospho-proteome via the Mitigation of Peptide-Metal Interactions.

Liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) has become the default platform for proteomics due to its specificity, sensitivity and sample compatibility. However, interactions between transition metals in LC systems and analytes containing phosphate groups result in poor chromatographic performance or even analyte loss. The use of systems where the metal surfaces had been treated with a hybrid inorganic/organic surface material to form an effective surface barrier mitigated these undesired interactions.

A comprehensive LFQ benchmark dataset on modern day acquisition strategies in proteomics.

In the last decade, a revolution in liquid chromatography-mass spectrometry (LC-MS) based proteomics was unfolded with the introduction of dozens of novel instruments that incorporate additional data dimensions through innovative acquisition methodologies, in turn inspiring specialized data analysis pipelines. Simultaneously, a growing number of proteomics datasets have been made publicly available through data repositories such as ProteomeXchange, Zenodo and Skyline Panorama. However, developing algorithms to mine this data and assessing the performance on different platforms is currently hampered by the lack of a single benchmark experimental design.

Epigenetic reprogramming enhances the therapeutic efficacy of osteoblast-derived extracellular vesicles to promote human bone marrow stem cell osteogenic differentiation.

Extracellular vesicles (EVs) are emerging in tissue engineering as promising acellular tools, circumventing many of the limitations associated with cell-based therapies. Epigenetic regulation through histone deacetylase (HDAC) inhibition has been shown to increase differentiation capacity. Therefore, this study aimed to investigate the potential of augmenting osteoblast epigenetic functionality using the HDAC inhibitor Trichostatin A (TSA) to enhance the therapeutic efficacy of osteoblast-derived EVs for bone regeneration.

Proteomic consequences of the deletion of cytochrome P450 (CYP450) reductase in mice.

Microsomal cytochrome P450 (CYP450) reductase enzymes play a major role in drug and xenobiotic metabolism. Mice which are deficient in hepatic CYP450 reductase serve as excellent models in understanding CYP450 drug metabolism and alterations in the underlying biology and function of these enzymes. A reversed-phase nano-bore UPLC-MS-based proteomic analysis, using an untargeted data independent approach (DIA), has been utilized for liver tissue extracts to evaluate differences between the proteomes of C57Bl6 wild type (WT) and hepatic P450 reductase mice (HRN™).

The Pharmacometabodynamics of Gefitinib after Intravenous Administration to Mice: A Preliminary UPLC-IM-MS Study.

The effects of intravenous gefitinib (10 mg/kg), an anilinoquinazoline thymidylate kinase inhibitor (TKI), selective for the epidermal growth factor receptor (EGFR), on the urinary metabotypes of mice were studied. We hypothesized that, in response to the administration of gefitinib, there might be significant changes in the excretion of many endogenous metabolites in the urine, which could be correlated with the plasma pharmacokinetics (PK) of the drug. In order to investigate this conjecture, urine from male C57 BL6 mice was collected before IV dosing (10 mg/kg) and at 0-3, 3-8, and 8-24 h post-dose.

Hybrid organic/inorganic hybrid surface technology for increasing the performance of LC/MS(MS)-based drug metabolite identification studies: Application to gefitinib and metabolites in mouse plasma and urine.

The detection, identification and quantification of drug metabolites plays a key role in drug discovery and development. Liquid chromatography (LC) coupled to mass spectrometry (MS) has become the primary technology for these studies due to its sensitivity and specificity. However, the presence of transition metals in the chromatography system and columns can result in non-specific and unwanted interactions with the drug and/or its metabolites, via electron-pair donation, leading to poor chromatography and analyte loss.

Mass spectrometry-based metabolomics for the discovery of candidate markers of flavonoid and polyphenolic intake in adults.

Robust biological markers of dietary exposure are essential in improving the understanding of the link between diet and health outcomes. Polyphenolic compounds, including flavonoids, have been proposed to mitigate the risk of chronic diseases where oxidative stress and inflammation play a central role. Biomarkers can provide objective measurement of the levels of polyphenolic compounds.

High-Throughput Microbore Ultrahigh-Performance Liquid Chromatography-Ion Mobility-Enabled-Mass Spectrometry-Based Proteomics Methodology for the Exploratory Analysis of Serum Samples from Large Cohort Studies.

The deployment of proteomic analysis in clinical studies represents a significant opportunity to detect and validate biomarkers in translational medicine, improve disease understanding, and provide baseline information on population health. However, comprehensive proteome studies usually employ nanoscale chromatography and often require several hours of analysis/sample. Here, we describe a high-throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) methodology using 1 mm scale chromatography requiring only 15 min/sample, coupled to ion mobility-enabled mass spectrometry.