MS-based Approaches Research Articles

Combining the Strengths of MS and NMR in Biochemometrics: A Case Study on Buddleja officinalis.

Biochemometrics has emerged as promising strategy for the targeted identification of bioactive constituents from natural sources. It is based on the correlation of bioactivity data with chemical data to reveal constituents contributing to activity. Providing complementary data and structural information, MS- and NMR-based biochemometric approaches have both been separately applied in the past. The herein presented study is dedicated to the evaluation of a combined MS- and NMR-based biochemometric workflow for the unambiguous identification of bioactives. As an example, a flower extract of Buddleja officinalis Maxim. was selected to unravel bioactive constituents in the context of dry eye disease pathology. While NMR-based biochemometrics relies on heterocovariance analysis (HetCA) of 1H NMR spectra using the previously established ELINA approach, a biochemometric molecular network was generated for the MS-based approach. Both analyses were performed in parallel and were ultimately combined to increase their power to identify the bioactive constituents from the complex mixture. As a result, phenylethanoid glycosides and triterpene saponins were discovered as main contributors for the antioxidant and cytotoxic effects of the extract. This article illustrates the advantages, opportunities, and limitations of MS and NMR in the context of biochemometrics.

IsoAsp-Quest: Workflow development for isoAsp identification using database searches.

A recent study reported that isomerization of aspartyl residues (Asp) occurs in various tissues and proteins in vivo. For a comprehensive analysis of post-translational modifications, the MS-based proteomic approach is a straightforward method; however, the isomerization of Asp does not alter its molecular weight. Therefore, a unique method is required to analyze Asp isomers using mass spectrometry. Herein, we present a novel strategy, isoAsp-Quest, which is a database search-oriented isoAsp identification method. isoAsp is specifically converted to 18O-labeled Lα-Asp by the enzymatic reaction of protein L-isoaspartyl-O-methyltransferase (PIMT) in 18O water with a mass shift of 2 Da, which, in principle, enables us to distinguish Asp isomers. However, in practice, a labeled Lα-Asp signal overlaps with that of endogenous Lα-Asp, making detection challenging. Therefore, degradation of the endogenous Lα-Asp peptide by AspN and subsequent removal of AspN were performed prior to the PIMT reaction. This strategy was applied to bovine lens α-crystallin. Consequently, several Asp isomerization sites, consistent with human αA-crystallin, were identified in bovine αA-crystallin, indicating that this strategy is also effective for biological proteins. Therefore, isoAsp-Quest enables the analysis of Lβ-Asp in a straightforward and rapid workflow, which may be useful for the quality control of protein products and biomarker discovery.

Spatial distribution of metabolites in processing Ziziphi Spinosae Semen as revealed by matrix-assisted laser desorption/ionization mass spectrometry imaging

Ziziphi Spinosae Semen (ZSS) is the first choice for the treatment of insomnia. This research aimed to reveal the spatial distribution of identifying quality markers of ZSS and to illustrate the metabolite quality characteristics of this herbal medicine. Here, we performed a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in situ to detect and image 33 metabolites in ZSS, including three saponins, six flavonoids, four alkaloids, eight fatty acids, and 12 amino acids. The MALDI images of the metabolites clearly showed the heterogeneous spatial distribution in different regions of ZSS tissues, such as the cotyledon, endosperm, and radicle. The distribution area of two saponins, six flavonoids, and three alkaloids increased significantly after the fried processing of ZSS. Based on the ion images, samples with different processing technologies were distinguished unambiguously by the pattern recognition method of orthogonal partial least squares discrimination analysis (OPLS-DA). Simultaneously, 23 major influencing components exerting higher ion intensities were identified as the potential quality markers of ZSS. Results obtained in the current research demonstrate that the processing of ZSS changes its content and distribution of the medicinal components. The analysis of MALDI-MSI provides a novel MS-based molecular imaging approach to investigate and monitor traditional medicinal plants.

Untargeted Metabolomics of the Sea Cucumber, Bohadschia marmorata (Jaeger, 1833)

The marine invertebrates such as sea cucumber are regarded as sources of diverse bioactive compounds with promising health and nutritional benefits. Many commercially traded sea cucumber species have been explored, yet none of the local low-value types in the province of La Union, Philippines. This study aimed to establish a baseline metabolomic profile of Bohadschia marmorata (Jaeger, 1833). The identification of compounds in its body wall utilized an Orbitrap MS-based approach. Metabolites were annotated using Compound Discover 3.2, and similarity searches were performed from online databases. Eleven putatively identified compounds, which included saponin (3beta,5xi,9xi)-28-Hydroxy-28-oxoolean-12-en-3-yl-beta-Dgalac topyranosyl-(1-3)-[beta-D-glucopyranosyl-(1-2)]-beta-D-glucopyranosiduronic acid, fatty acids and their byproducts such as eicosanoid (11,12-Epoxy-(5Z,8Z,11Z)-icosatrienoic acid) and prostanoids (13,14-dihydro Prostaglandin F1?; 13,14-Dihydro-15-keto Prostaglandin A2), dicarboxylic acids (tetradecanedioic acid; dodecanedioic acid; suberic acid; NP-001596), and amino acid (4-Oxoproline). The metabolic profile of the B. marmorata body wall showed a diverse group of representative metabolites common among holothurians associated with innate defense mechanisms, structural composition, and products of metabolic pathways. There is, however, a need to further isolate these compounds using other methods and test their biological activities.

Anion Exchange Chromatography-Mass Spectrometry to Characterize Proteoforms of Alpha-1-Acid Glycoprotein during and after Pregnancy.

Alpha-1-acid glycoprotein (AGP) is a heterogeneous glycoprotein fulfilling key roles in many biological processes, including transport of drugs and hormones and modulation of inflammatory and immune responses. The glycoform profile of AGP is known to change depending on (patho)physiological states such as inflammatory diseases or pregnancy. Besides complexity originating from five N-glycosylation sites, the heterogeneity of the AGP further expands to genetic variants. To allow in-depth characterization of this intriguing protein, we developed a method using anion exchange chromatography (AEX) coupled to mass spectrometry (MS) revealing the presence of over 400 proteoforms differing in their glycosylation or genetic variants. More precisely, we could determine that AGP mainly consists of highly sialylated higher antennary structures with on average 16 sialic acids and 0 or 1 fucose per protein. Interestingly, a slightly higher level of fucosylation was observed for AGP1 variants compared to that of AGP2. Proteoform assignment was supported by integrating data from complementary MS-based approaches, including AEX-MS of an exoglycosidase-treated sample and glycopeptide analysis after tryptic digestion. The developed analytical method was applied to characterize AGP from plasma of women during and after pregnancy, revealing differences in glycosylation profiles, specifically in the number of antennae, HexHexNAc units, and sialic acids.

Abstract PO2-14-11: Proteogenomic approaches for the identification of NF1/neurofibromin-depleted estrogen receptor positive breast cancers for targeted treatment

Abstract NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader (SERD), together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER+ models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors. We examined a panel of ER+ PDX models by DNA and mRNA sequencing and found that more than half of these models carried an NF1 shallow deletion and generally have low mRNA levels. Consistent with RAS and ER activation, RET and MEK levels in NF1-depleted tumors were elevated when profiled by mass spectrometry (MS) after kinase inhibitor bead pull-down. MS showed that NF1 can also directly and selectively bind to palbociclib-conjugated beads, aiding quantification. An immunohistochemistry (IHC) assay was also established to measure NF1, but the MS-based approach was more quantitative. Combined IHC and MS analysis defined a threshold of NF1 protein loss in ER+ breast PDX, below which tumors regressed upon treatment with B+F. These results suggest that we now have a MS-verified NF1 IHC assay that can be used for patient selection as a complement to somatic genomic analysis. Citation Format: Beom-Jun Kim, Ze-Yi Zheng, Jonathan Lei, Matthew Holt, Anran Chen, Jianheng Peng, Diana Fandino, Purba Singh, Hilda Kennedy, Yongchao Dou, M Rosario Chica-Parrado, Emmanuel Bikorimana, Dan Ye, Yunguan Wang, Ariella Hanker, Nada Mohamed, Susan Hilsenbeck, Bora Lim, Jaya Ruth Asirvatham, Arun Sreekumar, Bing Zhang, George Miles, Meenakshi Anurag, Matthew Ellis, Eric Chang. Proteogenomic approaches for the identification of NF1/neurofibromin-depleted estrogen receptor positive breast cancers for targeted treatment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-11.

In-Depth Proteome Coverage of In Vitro-Cultured Treponema pallidum and Quantitative Comparison Analyses with In Vivo-Grown Treponemes.

Previous mass spectrometry (MS)-based global proteomics studies have detected a combined total of 86% of all Treponema pallidum proteins under infection conditions (in vivo-grown T. pallidum). Recently, a method was developed for the long-term culture of T. pallidum under in vitro conditions (in vitro-cultured T. pallidum). Herein, we used our previously reported optimized MS-based proteomics approach to characterize the T. pallidum global protein expression profile under in vitro culture conditions. These analyses provided a proteome coverage of 94%, which extends the combined T. pallidum proteome coverage from the previously reported 86% to a new combined total of 95%. This study provides a more complete understanding of the protein repertoire of T. pallidum. Further, comparison of the in vitro-expressed proteome with the previously determined in vivo-expressed proteome identifies only a few proteomic changes between the two growth conditions, reinforcing the suitability of in vitro-cultured T. pallidum as an alternative to rabbit-based treponemal growth. The MS proteomics data have been deposited in the MassIVE repository with the data set identifier MSV000093603 (ProteomeXchange identifier PXD047625).

Metabolomics combined with chemometric analysis to identify α-glucosidase inhibitors in Phaleria macrocarpa fruit extracts and its molecular docking simulation

Phaleria macrocarpa is a medicinal plant widely available in Malaysia. The plant parts have been traditionally used as an antidiabetic remedy. This study aimed to identify the putative inhibitors of the α-glucosidase enzyme from P. macrocarpa using a gas chromatography-mass spectrometry (GC–MS)-based metabolomics approach and further subjected them to in silico molecular docking analysis to elucidate the possible mechanism of action. This study assessed the inhibitory potential of P. macrocarpa fruit extracts (aqueous, 20 %, 40 %, 60 %, 80 %, and 100 % ethanol) against the α-glucosidase enzyme using GC–MS and chemometric analysis. Orthogonal partial least squares (OPLS) combined with GC–MS analysis were applied to correlate the inhibition of enzyme activity to the metabolites profile of P. macrocarpa. All the potential inhibitors identified were further docked to the yeast (Saccharomyces cerevisiae) protein crystal structure (PDB3A4A). The generated score scatter plot of OPLS showed a recognizable separation of all the extracts into six different clusters. GC–MS, incorporated with multivariate data analysis techniques, was used to identify significant chemical markers. Methyl α-d-glucopyranoside, squalene, palmitic acid, myo-inositol and isoquinoline metabolites were identified as putative inhibitors against α-glucosidase activity from P. macrocarpa. In conclusion, the GC–MS-based metabolomics approach identified potential chemical markers of P. macrocarpa that could be utilized in the development of herbal based medicine.

Chemometric guided isolation of new triterpenoid saponins as acetylcholinesterase inhibitors from seeds of Achyranthes bidentata Blume

Achyranthes bidentata Blume (Amaranthaceae) is an annual or perennial herb widely used as ethnomedicine in Traditional Chinese Medicine for treating fever, cold, ulcers, mensural pain, dementia, and osteoporosis. In the current study, UPLC-IM-Q-TOF-MS/MS-based chemometric approach was adopted for the tentative identification of fifty-six compounds in the extract and fractions of A.bidentata seeds. Further, the chemometric-guided isolation led to the isolation of two previously undescribed oleanane-type triterpenoid saponins, named achyranosides A-B (27 and 30), along with three known compounds (31, 44, and 23) from water fraction of A. bidentata seeds. The structures of new compounds were elucidated based on the detailed analysis of NMR, HR-ESI-MS, FT-IR spectral data, and GC-FID techniques. The isolated compounds in vitro acetylcholinesterase inhibitory activity revealed the promising activity of chikusetsusaponin IVa (23) (IC50 = 63.7 μM) with mixed type of AChE inhibition in enzyme kinetic studies. Additionally, in silico binding free energy of isolated compounds disclosed the greater stability of enzyme-ligand complex owing to underlying multiple H-bond interactions. Overall, the study demonstrates the effectiveness of a chemometric-guided approach for the phytochemical exploration and isolation of new oleanane-type triterpenoid saponins from A. bidentata seeds.

Pathogenicity of monokaryotic and dikaryotic mycelia of Ganoderma boninense revealed via LC–MS-based metabolomics

This study compared the pathogenicity of monokaryotic (monokaryon) and dikaryotic (dikaryon) mycelia of the oil palm pathogen Ganoderma boninense via metabolomics approach. Ethyl acetate crude extracts of monokaryon and dikaryon were analysed by liquid chromatography quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF–MS) coupled with multivariate data analysis using MetaboAnalyst. The mummichog algorithm was also used to identify the functional activities of monokaryon and dikaryon without a priori identification of all their secondary metabolites. Results revealed that monokaryon produced lesser fungal metabolites than dikaryon, suggesting that monokaryon had a lower possibility of inducing plant infection. These findings were further supported by the identified functional activities. Monokaryon exhibits tyrosine, phenylalanine, and tryptophan metabolism, which are important for fungal growth and development and to produce toxin precursors. In contrast, dikaryon exhibits the metabolism of cysteine and methionine, arginine and proline, and phenylalanine, which are important for fungal growth, development, virulence, and pathogenicity. As such, monokaryon is rendered non-pathogenic as it produces growth metabolites and toxin precursors, whereas dikaryon is pathogenic as it produces metabolites that are involved in fungal growth and pathogenicity. The LC–MS-based metabolomics approach contributes significantly to our understanding of the pathogenesis of Ganoderma boninense, which is essential for disease management in oil palm plantations.

Quantitative profiling of posttranslational modifications of pathological tau via sarkosyl fractionation and mass spectrometry.

Tau protein aggregation is associated with posttranslational modifications (PTMs) in 75% of all dementia cases. The distribution of tau pathology and the presence of specific tau phosphorylation sites of interest are typically visualized and measured using antibodies. However, previous knowledge of the target epitopes is required. Additionally, antibodies can be used in a semi-quantitative manner but cannot be used to determine the absolute amount of tau or the extent of the modifications at specific sites or domains. Here we present a discovery assay that characterizes the global qualitative and quantitative tau modification landscape of a sample without a priori knowledge. Our workflow uses sarkosyl fractionation to extract the pathological tau species from sample-limited brain specimens, followed by mass spectrometry (MS) to characterize and quantify tau PTMs. The two-step MS-based proteomics approach includes an exploratory tau PTM analysis and a targeted full-length expressed stable isotope-labeled tau assay, which monitors specific unmodified tau peptides using a heavy isotope-labeled internal standard as a reference. This enables the absolute quantification of the respective tau peptides and the total tau amount in the sample, thus providing the modification extent of tau PTMs. This approach provides precise, comprehensive, qualitative and quantitative tau PTM profiling of the sample. It also enables the detailed molecular comparison of tau across multiple experiments, including a comparison between neurodegenerative diseases, stages of the disease, human patient heterogeneity and characterization of animal models. The approach is useful for studying the molecular features of pathological tau in neurodegeneration. The procedure requires 7-8 d and is suitable for users with expertise in targeted and untargeted MS-based protein analysis.

Mining Secondary Metabolites of Chassalia curviflora Leaves Using SIRIUS and High-Resolution Mass Spectrometry Data

Chassalia curviflora (Wall ex Kurz.) Thwaites is a flowering plant from Rubiaceae family. Previous phytochemical investigation on Chassalia species led to the isolation of benzoic acid, benzoquinone derivatives, flavonoids, monoterpenoid indole alkaloids and its essential oil composition. However, these are still scarce in terms of its scientific report on studied species. This study was carried out to investigate fast screening of secondary metabolites by using data-dependent acquisition (DDA) LC-MS/MS analysis of methanolic extract of the leaf of the C. curviflora in positive ionization mode. The data were analysed using computational mass spectrometry method from molecular structure database search implemented in Sirius. This led in discovering of 13 known secondary metabolites from a distinct tandem MS data. De novo molecular formula annotations and predictions from SIRIUS suggested five major class compounds classified as coumarin, benzofuran, fatty acids, esters and triterpenes. Stigmasterol 12 and β-sitosterol 13 were successfully isolated, thus used to verify the data obtained from the Sirius. With our results, we advocate the MS-based approach as a useful starting method for the dereplication of compounds from the complex crude extract of plants.

Blood-Derived Lipid and Metabolite Biomarkers in Cardiovascular Research from Clinical Studies: A Recent Update.

The primary prevention, early detection, and treatment of cardiovascular disease (CVD) have been long-standing scientific research goals worldwide. In the past decades, traditional blood lipid profiles have been routinely used in clinical practice to estimate the risk of CVDs such as atherosclerotic cardiovascular disease (ASCVD) and as treatment targets for the primary prevention of adverse cardiac events. These blood lipid panel tests often fail to fully predict all CVD risks and thus need to be improved. A comprehensive analysis of molecular species of lipids and metabolites (defined as lipidomics and metabolomics, respectively) can provide molecular insights into the pathophysiology of the disease and could serve as diagnostic and prognostic indicators of disease. Mass spectrometry (MS) and nuclear magnetic resonance (NMR)-based lipidomics and metabolomics analysis have been increasingly used to study the metabolic changes that occur during CVD pathogenesis. In this review, we provide an overview of various MS-based platforms and approaches that are commonly used in lipidomics and metabolomics workflows. This review summarizes the lipids and metabolites in human plasma/serum that have recently (from 2018 to December 2022) been identified as promising CVD biomarkers. In addition, this review describes the potential pathophysiological mechanisms associated with candidate CVD biomarkers. Future studies focused on these potential biomarkers and pathways will provide mechanistic clues of CVD pathogenesis and thus help with the risk assessment, diagnosis, and treatment of CVD.

The Plasma Proteomic Landscape of Thrombotic Events across Multi-Center Cohorts

Background: Diagnosis and risk prediction of thrombosis is challenging due to nonspecific symptoms, diverse risk factors, symptom overlap with other conditions and the variability in thrombosis presentations. Little is known on the impact of thrombosis on the plasma proteome across the spectrum of its clinical representations. Objective: The aim of this study was to explore the plasma proteomic landscape across multi-center thrombosis cohorts. The focus was on identifying protein signatures representative of thrombosis in general or among patients at risk of thrombosis, those with a history of thrombosis, those at acute phase and those with COVID-19 associated thrombosis. Method: Using state-of-the-art mass spectrometry-based proteomics, we analyzed 420 plasma proteome samples both cross-sectional and longitudinal from 211 patients, including those at risk of thrombosis, in the acute thrombotic phase (cerebral venous sinus thrombosis patients) and with COVID-19-associated thrombosis (Figure 1). Linear models and unsupervised co-expression clustering were used to explore common and distinct protein signatures among the cohorts. Results: Across the analyzed samples 475 proteins were quantified, with 86% showing a CV below 30%, and we found a strong correlation between protein levels quantified by MS and ELISA. Plasma proteomic alterations exhibited variation across cohorts, with the most pronounced changes observed in COVID-19 patients. While co-expression analysis unveiled distinct protein clusters associated with SARS-CoV-2 signaling pathways (CRP, SAA1) and Vitamin K-dependent (VKA) coagulation proteins (F9, F2, F10, PROS, PROC, PROZ, C4BPA), we did not identify a specific protein cluster related to thrombosis (Figure 2). Notably, VKA-therapy was associated with decreased abundance of VKA-dependent coagulation proteins, with F10 and PROZ showing significant alterations between patients who experienced a thrombosis compared to healthy controls. We observed an inverse correlation between the International Normalized Ratio and abundance of VKA-dependent coagulation proteins, with an increased correlation of F2 and F9 in patients who experienced a thrombotic event. Additionally, our analysis also highlighted potential variations in multi-center cohorts during sample handling, emphasizing the importance of standardized protocols in ensuring the reliability and comparability of results. Conclusion: Using a comprehensive MS-based proteomics approach, we identified clear plasma proteomic signatures associated with VKA-treatment and COVID-19 , but no distinct thrombosis signature across multi-center thrombosis cohorts.

Unveiling the potential of proteomics in addressing food and feed safety challenges.

The food and feed sector in Europe is rapidly evolving to address contemporary challenges, striving for fairer, safer, greener and more sustainable food systems. This includes the exploration of new protein sources for human consumption and animal feed such as protein derived from insects, algae or novel plant-derived proteins, and the re-evaluation of existing sources like processed animal protein (PAP). To generate reliable data on the diverse array of emerging protein sources for future food and feed safety assessments, a growing demand for the development and implementation of advanced analytical techniques exists. New approach methodologies (NAMs) including, mass spectrometry (MS)-based proteomics methods have been emerging as valuable techniques which potentially can be implemented in regulatory laboratory settings to complement conventional approaches in this realm. These MS-driven strategies have already proven their utility in diverse applications, including the detection of prohibited substances in feed, identification of allergens, differentiation of fish species in complex mixtures for fraud detection and the verification of novel foods and alternative protein sources. This EU-FORA programme was focused on three core objectives namely: (i) the training of the fellow in utilising MS-based proteomics for food and feed safety analyses, (ii) the involvement of the fellow in the development of standardised operating procedures (SOP) for targeted and non-targeted proteomic MS-based workflows for species and tissues specific PAP identification in a national reference laboratory (NRL) and (iii) the transfer and implementation of MS-based approaches and standardised protocols for PAP analysis at the fellow's home institution. Altogether, this programme facilitates the broadening and diversification of use of MS-based proteomic methodologies for reinforcing their significance within the domains of food and feed safety research and regulatory science applications.

Characterizing citrullination by mass spectrometry-based proteomics.

Citrullination is an important post-translational modification (PTM) of arginine, known to play a role in autoimmune disorders, innate immunity response and maintenance of stem cell potency. However, citrullination remains poorly characterized and not as comprehensively understood compared to other PTMs, such as phosphorylation and ubiquitylation. High-resolution mass spectrometry (MS)-based proteomics offers a valuable approach for studying citrullination in an unbiased manner, allowing confident identification of citrullination modification sites and distinction from deamidation events on asparagine and glutamine. MS efforts have already provided valuable insights into peptidyl arginine deaminase targeting along with site-specific information of citrullination in for example synovial fluids derived from rheumatoid arthritis patients. Still, there is unrealized potential for the wider citrullination field by applying MS-based mass spectrometry approaches for proteome-wide investigations. Here we will outline contemporary methods and current challenges for studying citrullination by MS, and discuss how the development of neoteric citrullination-specific proteomics approaches still may improve our understanding of citrullination networks. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Direct Competition of ATCUN Peptides with Human Serum Albumin for Copper(II) Ions Determined by LC-ICP MS.

Copper is an indispensable biometal, primarily serving as a redox-competent cofactor in numerous proteins. Apart from preformed copper-binding sites within the protein structures, small peptide motifs exist called ATCUN, which are composed of an N-terminal tripeptide XZH, able to bind Cu(II) ions in exchangeable form. These motifs are common for serum albumin, but they are also present in a wide range of proteins and peptides. These proteins and peptides can be involved in copper metabolism, and copper ions can affect their biological role. The distribution of copper between the ATCUN peptides, including truncated amyloid-β (Aβ) peptides Aβ4-42 and Aβ11-42, which may be involved in Alzheimer's disease pathogenesis, is mainly determined by their concentrations and relative Cu(II)-binding affinities. The Cu(II)-binding affinity (log Kd) of several ATCUN peptides, determined by different methods and authors, varies by more than three orders of magnitude. This variation may be attributed to the chemical properties of peptides but can also be influenced by the differences in methods and experimental conditions used for the determination of Kd. In the current study, we performed direct competition experiments between selected ATCUN peptides and HSA by using an LC-ICP MS-based approach. We demonstrated that ATCUN and truncated Aβ peptides Aβ4-16 and Aβ11-15 bind Cu(II) ions with an affinity similar to that for HSA. Our results demonstrate that ATCUN motifs cannot compete with excess HSA for the binding of Cu(II) ions in the blood and cerebrospinal fluid.

Discovery and Characterization of an Acid-Labile Serine-Lysine Cross-Link in Antibody High-Molecular-Weight Species Using a Multipronged Mass Spectrometry Approach.

Characterizing the cross-links responsible for the covalent high-molecular-weight (HMW) species in therapeutic monoclonal antibodies (mAbs) is of great importance as it not only provides a framework for risk assessment but also offers insights for process improvement. However, owing to the complexity and low abundance, identification of novel and unknown cross-links in mAb products can be very challenging. Here, applying a multipronged MS-based approach, we report the discovery of a novel covalent cross-link formed via an imine bond between lysine and serine residues. In particular, this Ser-Lys cross-link was found to be acid-labile and can be easily overlooked by conventional LC-MS techniques operated at low pH. It is worth noting that although imine-based cross-link has been previously reported in collagen protein cross-linking, this is the first time that a Ser-Lys cross-link has been found in a mAb product that contributes to covalent HMW species formation.

LC-MS/MS-based targeted amino acid metabolic profile of Auricularia cornea grown on pinecone substrate

Pinecone substrate offers an eco-friendly and cost-effective alternative for cultivating edible fungi. This pioneering study explores the 94 amino acids metabolic profiles of Auricularia cornea grown on various pinecone substrates. To our knowledge, this is the first study of quantify A. cornea on an oleaginous substrate (pinecone) using targeted LC-MS /MS-based metabolomics approaches. Five different pinecone substrate percentages (0%, 7%, 14%, 21%, and 28% respectively) were used for A. cornea culture, and the resulting fruiting bodies were analyzed for amino acids metabolic profiles. Detected 79 amino acids metabolites, 15 undetected. High contents of succinic-acid and γ-aminobutyric acid. Thirty-three amino acid metabolites showed significant differences between groups, primarily related to protein synthesis. KEGG analysis revealed that seven major metabolic pathways were significantly enriched. The findings provide valuable insights into the metabolite composition of A. cornea grown on a pinecone substrate, potentially contribute to the understanding of its nutritional and medicinal properties.

Influence of drought stress on increasing bioactive compounds of pomegranate (Punica granatum L.) juice. Exploratory study using LC–MS-based untargeted metabolomics approach

Water deficit is one of the most important abiotic factors that reduce the agricultural productivity. The impact of irrigation levels on bioactive compounds of one commercial pomegranate cultivar (‘Mollar de Elche’) was studied through LC–MS-based untargeted metabolomics approach. Two treatments were applied to explore crop responses to the timing and the regime of the irrigation applications. The main goal of the present study was to validate the untargeted metabolomics approach as analytical tool to find new markers related with the water deficit. Robust classification and prediction model was built with the use of supervised technique, partial least squares discriminant analysis (PLS-DA). The results revealed that pomegranate juices obtained from plants grown under DIr conditions experimented physiological stress, which significantly increased their bioactive compounds (polyphenols, phenylpropanoids, peptides, benzoic acid, tannins, and phospholipids) in comparison with the Control treatments. The results indicate that controlled irrigation stress would increase the production of bioactive compounds by increasing the phenylpropanoids metabolism. New metabolites were tentatively identified in pomegranate juice by the untargeted metabolomics techniques. This study showed, for the first time, the satisfactory application of the untargeted metabolomics approach using an UHPLC-QTOF system to identify bioactive secondary metabolites from pomegranate juices to discriminate among different irrigation conditions.