•A PRM-based targeted lipidomics workflow was proposed on Q-TOF MS for the quantification of 222 lipids.•This PRM-based quantification strategy provide high quality MS/MS spectra for both identification and quantification.•A software solution was proposed to use multiple product ions for quantification, improving the reliability.•The developed workflow was applied to discovery perturbed lipids in serum between different disease stages of SLE.Advances in high-resolution mass spectrometers with faster scanning capabilities and higher sensitivities have expanded these instruments' functionality beyond traditional data-dependent acquisition in targeted metabolomics. Apart from the traditional multiple reaction monitoring strategy, the parallel reaction monitoring (PRM) strategy is also used for targeted metabolomics quantification. The high resolution and mass accuracy of full-scan (MS1) and tandem mass spectrometry (MS/MS) scan result in sufficient selectivity by monitoring all MS/MS fragment ions for each target precursor and simultaneously providing flexibility in assay method construction and post-acquisition data analysis. In this study, using an orthogonal quadrupole-time of flight liquid chromatography-mass spectrometry system (QTOF LC-MS), we investigated the applicability of a large-scale targeted lipidomic assay using scheduled PRM. This method monitored 222 lipids belonging to 15 lipid species in serum. Robustness, reproducibility, and quantitative performance were assessed using chemical standards and serum samples. Finally, we demonstrated the application of this PRM-based targeted metabolomic workflow to systemic lupus erythematosus, a severe autoimmunological disease. Results showed that 63 lipids belonging to 11 lipid species were significantly changed. In summary, at the first time, a robust targeted lipidomic workflow was established using PRM acquisition strategy on a Q-TOF platform, providing another powerful tool for targeted metabolomic analysis.Download high-res image (191KB)Download full-size image

•First method for determination of glycine-β-muricholic acid (Gly-MCA) in mice plasma by HPLC–MS/MS.•This HPLC–MS/MS method is simple, rapid and sensitive, with 5.0 min runtime per sample.•The developed method was successfully applied to the pharmacokinetic study of Gly-MCA in mice.A simple, sensitive and rapid method using high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) was developed and validated for quantification of glycine-β-muricholic acid (Gly-MCA) in mouse plasma for the first time. Plasma samples were pretreated with protein precipitation. The analyte and internal standard were separated on a Shimadzu Shim-pack XR-ODS column (4.6 × 50 mm, 2.2 μm) using 0.1% formic acid-water-methanol as mobile phase, with a runtime of 5 min. Detection was performed using negative ion electrospray tandem mass spectrometry via multiple reaction monitoring (MRM) scan mode. The linear range was 5 ng–2 μg/ml (correlation coefficient >0.995) for Gly-MCA with a lower limit of quantitation of 5 ng/ml. The intra-day and inter-day precision were less than 9.2% for the analyte and accuracy was from 0.4% to 7.0%. This analytical method was then successfully applied to a pharmacokinetic study of Gly-MCA following oral administration and intraperitoneal injection in mice.

•DIA workflow is constructed on Q-Orbitrap platform for metabolomics analysis.•HILIC/RPLC-DIA-MS workflows are constructed for analysis of metabolites and lipids.•Performances of DDA, DIA, and AIF strategies were compared and discussed.•Serum of the thyroid carcinoma patients is analyzed by DIA-MS to reveal perturbed metabolites.Untargeted metabolomic profiling has been widely used in recent years. However, the low reproducibility of the data-dependent acquisition (DDA) strategy presents a major bottleneck that considerably limits the reliability of metabolomic studies in biological and clinical research. The data-independent acquisition (DIA) strategy is proposed to solve the above-mentioned problem, and it is gaining popularity. This paper presents a novel approach for performing metabolomic analysis using an untargeted, liquid chromatography–data independent-mass spectrometry (LC-DIA-MS) strategy on a quadrupole-Orbitrap platform. Using chemical standards and metabolites extracted from serum samples, we optimized the LC-DIA-MS parameters to analyze hydrophilic metabolites and lipids. The quantitative performance and analytical reliability were evaluated, and the performances of DIA, DDA, and all-ion fragmentation mode were compared. Finally, as a proof of concept, we applied the constructed DIA workflow to a comparative metabolomic study of papillary thyroid carcinoma (TC) serum samples. Several metabolites, including carnitine, trimethylamine N-oxide, and some amino acids, significantly changed between patients and healthy controls. This study demonstrated the feasibility and advantage of the DIA strategy on untargeted metabolomic analysis for biological study and clinical biomarker screening.

Recent advances in mass spectrometers which have yielded higher resolution and faster scanning speeds have expanded their application in metabolomics of diverse diseases. Using a quadrupole-Orbitrap LC–MS system, we developed an efficient large-scale quantitative method targeting 237 metabolites involved in various metabolic pathways using scheduled, parallel reaction monitoring (PRM). We assessed the dynamic range, linearity, reproducibility, and system suitability of the PRM assay by measuring concentration curves, biological samples, and clinical serum samples. The quantification performances of PRM and MS1-based assays in Q-Exactive were compared, and the MRM assay in QTRAP 6500 was also compared. The PRM assay monitoring 237 polar metabolites showed greater reproducibility and quantitative accuracy than MS1-based quantification and also showed greater flexibility in postacquisition assay refinement than the MRM assay in QTRAP 6500. We present a workflow for convenient PRM data processing using Skyline software which is free of charge. In this study we have established a reliable PRM methodology on a quadrupole-Orbitrap platform for evaluation of large-scale targeted metabolomics, which provides a new choice for basic and clinical metabolomics study.

•Developing a HPLC–MS/MS method to simultaneously determining four compounds in metabolic pathway for phosphatidylcholine.•Matrix effects could be minimized by using stable deuterated isotope internal standards and matched matrix.•Levels of the four metabolites were compared between cocktail administered mice and controls.Trimethylamine-N-oxide (TMAO) is a metabolite generated from choline, betaine and carnitine in a gut microbiota-dependent way. This molecule is associated with development of atherosclerosis and cardiovascular events. A sensitive liquid chromatographic electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated for the simultaneous determination of TMAO related molecules including TMAO, betaine, choline, and carnitine in mouse plasma. Analytes are extracted after protein precipitation by methanol and subjected to LC-ESI-MS/MS without preliminary derivatization. Separation of analytes was achieved on an amide column with acetonitrile-water as the mobile phase. This method has been fully validated in this study in terms of selectivity, linearity, sensitivity, precision, accuracy, and carryover effect, and the stability of the analyte under various conditions has been confirmed. This developed method has successfully been applied to plasma samples of our mouse model.

•Effects of nucleotide sites in acid damage of DNA were studied with MALDI MS.•Terminal nucleotides were easily damaged than middle ones in MALDI MS.•5′-Terminal was easily damaged than 3′-ones in the further step in MALDI MS.Acid hydrolysis of DNA oligonucleotides leads to nucleotides damage, especially affecting those containing purine bases. Previous research focused mainly on the types of cleaved nucleobases and the cleavage efficiency. There has been no report about the nucleotide site. In this study, 10 DNA oligonucleotides with differing dAMP and dGMP sites were synthesized to study the influence of these sites with MALDI-TOF MS, which offers a powerful tool with advantages of rapid speed, high sensitivity, high tolerance and simple mass spectra. Results show that, with acid hydrolysis, cleavage of terminal nucleotides differs from those in the middle in the MALDI-TOF mass spectra. In addition, in the course of further degradation, there are obvious differences between nucleotides at the 5′ and 3′-termini.

In this study we describe a method for highly specific enrichment of glycopeptides with boronic acid-functionalized chitosan polymeric nanospheres and matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS). This is the first time chitosan has been used to create nanosphere support material for selective enrichment of glycopeptides by modification with glycidyl methacrylate (GMA) and derivatization with 3-aminophenylboronic acid (APB). Due to their multifunctional chemical moieties, these 20–100 nm chitosan–GMA–APB nanospheres have unique properties, such as good dispersibility, good biocompatibility and chemical stability, as well as augmented specificity with glycopeptides. Enrichment conditions were optimized by using trypsin digested glycoprotein horseradish peroxidase. The high specificity of chitosan–GMA–APB nanospheres was demonstrated by effectively enriching glycopeptides from a digest mixture of horseradish peroxidase and nonglycoproteins (bovine serum albumin (BSA)).Highlights► This report describes the highly specific capture of glycopeptides employing chitosan–GMA–APB nanospheres. ► These nanospheres have a diameter of 20–100 nm and were synthesized for this work. ► MALDI-TOF-MS combined with use of these nanospheres is a simple and rapid approach for enrichment of glycopeptides. ► The binding affinity of boronic acid and glycopeptides is pH and solvent dependent.

In this study, we describe characterization of the human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres. Chitosan-GMA-IDA-Cu(II) nanospheres with diameters of 20 to 100 nm have unique properties due to multifunctional chemical moieties, high surface area, high capacity, good dispersibility in buffer solution as well as good biocompatibility and chemical stability which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanospheres with MS spectrometry results in a novel strategy which should make it possible to characterize the plasma proteome in a single test. Peptides and proteins adsorbed on the nanosphere can be directly detected by MALDI-TOF-MS. The eluted lower molecular weight peptides and proteins are identified by nano-LC-ESI-MS/MS. A total of 842 unique LMW peptides and 1,682 human unredundant proteins IDs were identified in two different binding buffers, which included relatively low-level proteins (e.g., pg/mL of IL3 Interleukin-3) co-distributed with high-abundance proteins (e.g., 35–55 mg/mL level serum albumin). As such, this nanosphere technique selectively enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude. Considering this capacity for selective enrichment of peptides and proteins in human plasma, and the large number of LMW peptides and proteins which can be identified, this method promises to accelerate discovery of biomarkers for clinical application.