bims-mascan 2023-12-03 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2023–12–03
27 papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

CorrelationCalculator and Filigree: Tools for Data-Driven Network Analysis of Metabolomics Data.
Telomerase reverse transcriptase induces targetable alterations in glutathione and nucleotide biosynthesis in glioblastomas.
Multicenter Collaborative Study to Optimize Mass Spectrometry Workflows of Clinical Specimens.
Trace Sample Proteome Quantification by Data-Dependent Acquisition without Dynamic Exclusion.
A Bioconductor workflow for processing, evaluating, and interpreting expression proteomics data.
High-Throughput Comprehensive Quantitative LC-MS/MS Analysis of Common Drugs and Metabolites (62 Compounds) in Human Urine.
Quantitative LC-MS/MS Analysis of Amphetamines and Cocaine in Human Urine.
Adapting an Isobaric Tag-Labeled Yeast Peptide Standard to Develop Targeted Proteomics Assays.
Quantitative LC-MS/MS Analysis of Opiates and Opioids in Human Urine.
Adipose triglyceride lipase is a therapeutic target in advanced prostate cancer that promotes metabolic plasticity.
The immunometabolic ecosystem in cancer.
Quantification of Thiopurine Metabolites in Human Erythrocytes by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
Analysis of Barbiturates in Urine by LC-MS/MS.
Quantification of Olanzapine by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
A New, Rapid Method for the Quantification of Dolichyl Phosphates in Cell Cultures Using TMSD Methylation Combined with LC-MS Analysis.
A one-year pilot study comparing direct-infusion high resolution mass spectrometry based untargeted metabolomics to targeted diagnostic screening for inherited metabolic diseases.
Mass Spectrometry in Clinical Laboratory: Applications in Therapeutic Drug Monitoring and Toxicology.
CHRISTMAS: Chiral Pair Isobaric Labeling Strategy for Multiplexed Absolute Quantitation of Enantiomeric Amino Acids.
Quantitative Analysis of Buprenorphine, Norbuprenorphine, and Their Glucuronide Metabolites in Human Urine.
LC-MS Approaches for Oxysterols in Various Biosamples.
SpatialCells: Automated Profiling of Tumor Microenvironments with Spatially Resolved Multiplexed Single-Cell Data.
Novel approaches targeting ferroptosis in treatment of glioma.
Simultaneous separation and identification of all structural isomers and enantiomers of aminobutyric acid using a highly sensitive chiral resolution labeling reagent.
Tumor-derived extracellular vesicles and particles (EVPs): pivotal vectors in driving metabolic reprogramming.
Targeting the lactic acid metabolic pathway for antitumor therapy.
Comprehensive Overview of Bottom-up Proteomics using Mass Spectrometry.
Expression and Purification of Recombinant ADAMTS8.

J Vis Exp. 2023 Nov 10.

CorrelationCalculator and Filigree: Tools for Data-Driven Network Analysis of Metabolomics Data.

Gayatri Iyer, Marci Brandenburg, Christopher Patsalis, George Michailidis, Alla Karnovsky.

A significant challenge in the analysis of omics data is extracting actionable biological knowledge. Metabolomics is no exception. The general problem of relating changes in levels of individual metabolites to specific biological processes is compounded by the large number of unknown metabolites present in untargeted liquid chromatography-mass spectrometry (LC-MS) studies. Further, secondary metabolism and lipid metabolism are poorly represented in existing pathway databases. To overcome these limitations, our group has developed several tools for data-driven network construction and analysis. These include CorrelationCalculator and Filigree. Both tools allow users to build partial correlation-based networks from experimental metabolomics data when the number of metabolites exceeds the number of samples. CorrelationCalculator supports the construction of a single network, while Filigree allows building a differential network utilizing data from two groups of samples, followed by network clustering and enrichment analysis. We will describe the utility and application of both tools for the analysis of real-life metabolomics data.

DOI: https://doi.org/10.3791/65512
bioRxiv. 2023 Nov 16. pii: 2023.11.14.566937. [Epub ahead of print]

Telomerase reverse transcriptase induces targetable alterations in glutathione and nucleotide biosynthesis in glioblastomas.

Suresh Udutha, Céline Taglang, Georgios Batsios, Anne Marie Gillespie, Meryssa Tran, Sabrina M Ronen, Johanna Ten Hoeve, Thomas G Graeber, Pavithra Viswanath.

Telomerase reverse transcriptase (TERT) is essential for glioblastoma (GBM) proliferation. Delineating metabolic vulnerabilities induced by TERT can lead to novel GBM therapies. We previously showed that TERT upregulates glutathione (GSH) pool size in GBMs. Here, we show that TERT acts via the FOXO1 transcription factor to upregulate expression of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme of de novo GSH synthesis. Inhibiting GCLC using siRNA or buthionine sulfoximine (BSO) reduces synthesis of 13 C-GSH from [U- 13 C]-glutamine and inhibits clonogenicity. However, GCLC inhibition does not induce cell death, an effect that is associated with elevated [U- 13 C]-glutamine metabolism to glutamate and pyrimidine nucleotide biosynthesis. Mechanistically, GCLC inhibition activates MYC and leads to compensatory upregulation of two key glutamine-utilizing enzymes i.e., glutaminase (GLS), which generates glutamate from glutamine, and CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotatase), the enzyme that converts glutamine to the pyrimidine nucleotide precursor dihydroorotate. We then examined the therapeutic potential of inhibiting GLS and CAD in combination with GCLC. 6-diazo-5-oxy-L-norleucin (DON) is a potent inhibitor of glutamine-utilizing enzymes including GLS and CAD. The combination of BSO and DON suppresses GSH and pyrimidine nucleotide biosynthesis and is synergistically lethal in GBM cells. Importantly, in vivo stable isotope tracing indicates that combined treatment with JHU-083 (a brain-penetrant prodrug of DON) and BSO abrogates synthesis of GSH and pyrimidine nucleotides from [U- 13 C]-glutamine and induces tumor shrinkage in mice bearing intracranial GBM xenografts. Collectively, our studies exploit a mechanistic understanding of TERT biology to identify synthetically lethal metabolic vulnerabilities in GBMs.
SIGNIFICANCE: Using in vivo stable isotope tracing, metabolomics, and loss-of-function studies, we demonstrate that TERT expression is associated with metabolic alterations that can be synergistically targeted for therapy in glioblastomas.

DOI: https://doi.org/10.1101/2023.11.14.566937
J Proteome Res. 2023 Nov 28.

Multicenter Collaborative Study to Optimize Mass Spectrometry Workflows of Clinical Specimens.

Oliver Kardell, Christine von Toerne, Juliane Merl-Pham, Ann-Christine König, Marcel Blindert, Teresa K Barth, Julia Mergner, Christina Ludwig, Johanna Tüshaus, Stephan Eckert, Stephan A Müller, Stephan Breimann, Pieter Giesbertz, Alexander M Bernhardt, Lisa Schweizer, Vincent Albrecht, Daniel Teupser, Axel Imhof, Bernhard Kuster, Stefan F Lichtenthaler, Matthias Mann, Jürgen Cox, Stefanie M Hauck.

  The foundation for integrating mass spectrometry (MS)-based proteomics into systems medicine is the development of standardized start-to-finish and fit-for-purpose workflows for clinical specimens. An essential step in this pursuit is to highlight the common ground in a diverse landscape of different sample preparation techniques and liquid chromatography-mass spectrometry (LC-MS) setups. With the aim to benchmark and improve the current best practices among the proteomics MS laboratories of the CLINSPECT-M consortium, we performed two consecutive round-robin studies with full freedom to operate in terms of sample preparation and MS measurements. The six study partners were provided with two clinically relevant sample matrices: plasma and cerebrospinal fluid (CSF). In the first round, each laboratory applied their current best practice protocol for the respective matrix. Based on the achieved results and following a transparent exchange of all lab-specific protocols within the consortium, each laboratory could advance their methods before measuring the same samples in the second acquisition round. Both time points are compared with respect to identifications (IDs), data completeness, and precision, as well as reproducibility. As a result, the individual performances of participating study centers were improved in the second measurement, emphasizing the effect and importance of the expert-driven exchange of best practices for direct practical improvements.

Keywords:  CSF; LC–MS; R package mpwR; clinical specimen; data-dependent acquisition; data-independent acquisition; interlaboratory; intralaboratory; mass spectrometry; plasma; round-robin study

DOI:  https://doi.org/10.1021/acs.jproteome.3c00473
Anal Chem. 2023 Nov 30.

Trace Sample Proteome Quantification by Data-Dependent Acquisition without Dynamic Exclusion.

Ci Wu, Jiao Lei, Fei Meng, Xingyao Wang, Cassandra J Wong, Jiaxi Peng, Ge Lin, Anne-Claude Gingras, Junfeng Ma, Shen Zhang.

Despite continuous technological improvements in sample preparation, mass-spectrometry-based proteomics for trace samples faces the challenges of sensitivity, quantification accuracy, and reproducibility. Herein, we explored the applicability of turboDDA (a method that uses data-dependent acquisition without dynamic exclusion) for quantitative proteomics of trace samples. After systematic optimization of acquisition parameters, we compared the performance of turboDDA with that of data-dependent acquisition with dynamic exclusion (DEDDA). By benchmarking the analysis of trace unlabeled human cell digests, turboDDA showed substantially better sensitivity in comparison with DEDDA, whether for unfractionated or high pH fractionated samples. Furthermore, through designing an iTRAQ-labeled three-proteome model (i.e., tryptic digest of protein lysates from yeast, human, and E. coli) to document the interference effect, we evaluated the quantification interference, accuracy, reproducibility of iTRAQ labeled trace samples, and the impact of PIF (precursor intensity fraction) cutoff for different approaches (turboDDA and DEDDA). The results showed that improved quantification accuracy and reproducibility could be achieved by turboDDA, while a more stringent PIF cutoff resulted in more accurate quantification but less peptide identification for both approaches. Finally, the turboDDA strategy was applied to the differential analysis of limited amounts of human lung cancer cell samples, showing great promise in trace proteomics sample analysis.

DOI: https://doi.org/10.1021/acs.analchem.3c03357
F1000Res. 2023 ;12 1402

A Bioconductor workflow for processing, evaluating, and interpreting expression proteomics data.

Charlotte Hutchings, Charlotte S Dawson, Thomas Krueger, Kathryn S Lilley, Lisa M Breckels.

  Background: Expression proteomics involves the global evaluation of protein abundances within a system. In turn, differential expression analysis can be used to investigate changes in protein abundance upon perturbation to such a system. Methods: Here, we provide a workflow for the processing, analysis and interpretation of quantitative mass spectrometry-based expression proteomics data. This workflow utilizes open-source R software packages from the Bioconductor project and guides users end-to-end and step-by-step through every stage of the analyses. As a use-case we generated expression proteomics data from HEK293 cells with and without a treatment. Of note, the experiment included cellular proteins labelled using tandem mass tag (TMT) technology and secreted proteins quantified using label-free quantitation (LFQ). Results: The workflow explains the software infrastructure before focusing on data import, pre-processing and quality control. This is done individually for TMT and LFQ datasets. The application of statistical differential expression analysis is demonstrated, followed by interpretation via gene ontology enrichment analysis. Conclusions: A comprehensive workflow for the processing, analysis and interpretation of expression proteomics is presented. The workflow is a valuable resource for the proteomics community and specifically beginners who are at least familiar with R who wish to understand and make data-driven decisions with regards to their analyses.

Keywords:  Bioconductor; QFeatures; bottom-up proteomics; data processing; differential expression; limma; mass spectrometry; proteomics; quality control; shotgun proteomics

DOI:  https://doi.org/10.12688/f1000research.139116.1
Methods Mol Biol. 2024 ;2737 215-227

High-Throughput Comprehensive Quantitative LC-MS/MS Analysis of Common Drugs and Metabolites (62 Compounds) in Human Urine.

Putuma P Gqamana, Y Victoria Zhang.

  In recent years a multitude of LC-MS/MS assays have been widely reported in commercial and clinical literature demonstrating the simultaneous analyses of dozens of drugs of abuse in human samples. The utility of such assays is meant to supplant the indirect detection based on the classical spectral library approach. Direct and simultaneous analysis via LC-MS/MS technology is made possible by fast acquisition rates in multiple reaction monitoring, as well as sensitivity and high selectivity of the technology for each individual analyte in a complex mixture. Hence, unlike immunoassays, which are not well-suited for the analyses of mixtures, and which may also be prone to false positives from potential interferences, quantitative LC-MS/MS analyses are feasible for complex patient mixtures of drugs of abuse. We hereby present a robust clinical LC-MS/MS assay for the simultaneous and semi-quantitative analysis of up to 62 drugs of abuse in human urine, representing major classes that include opiates, benzodiazepines, amphetamines, etc. The assay utilizes dilute and shoot, whereby the sample is diluted ten times in internal standard reagent and thereafter submitted to the LC-MS instrument, i.e., reversed-phase liquid chromatography coupled to the electrospray ionization multiple reaction monitoring analysis, via the TSQ Endura triple-quadrupole instrument. The assay employs stable isotope-labeled internal standards with a linear response in the 30-300 ng/mL range, effectively semi-quantitative, since this analytical range is well within typical immunoassay cutoffs for most drugs.

Keywords:  Dilute and shoot; ESI-MRM-MS; Panel of drugs of abuse; Semi-quantitative

DOI:  https://doi.org/10.1007/978-1-0716-3541-4_20
Methods Mol Biol. 2024 ;2737 15-23

Quantitative LC-MS/MS Analysis of Amphetamines and Cocaine in Human Urine.

Putuma P Gqamana, Y Victoria Zhang.

  We hereby present a fast and high-throughput LC-MS/MS assay for the simultaneous analysis of amphetamines and cocaine in human urine. The assay is used for confirmations following immunoassay urine drug screens as well as a quantitative assay to report actual urine concentrations in the range 30-10,000 ng/mL for each of the seven analytes, namely, amphetamine; methamphetamine; phentermine; methylenedioxyamphetamine; 3,4-methylenedioxymethamphetamine; methylenedioxy-ethyl-amphetamine; and a cocaine metabolite, benzoylecgonine. The assay derives its efficacy from minimal sample preparation via dilute and shoot. The platform is based on reversed-phase liquid chromatography coupled to the TSQ Endura triple-quadrupole (QqQ) MS instrument for detection via electrospray ionization multiple-reaction monitoring MS. The quantitative analysis is based on the linear calibration whereby the instrument response for each analyte at a given concentration is normalized against stable isotope-labeled internal standard. In addition, the assay can be multiplexed across more than one LC channel to obtain high-sample throughput.

Keywords:  Amphetamines; Cocaine; Drugs of abuse; Electrospray ionization (ESI); High-throughput LC-MS/MS; Multiple-reaction monitoring (MRM); Multiplex HPLC

DOI:  https://doi.org/10.1007/978-1-0716-3541-4_2
J Proteome Res. 2023 Nov 27.

Adapting an Isobaric Tag-Labeled Yeast Peptide Standard to Develop Targeted Proteomics Assays.

Kevin D Dong, Ernst W Schmid, Ryan D Bomgarden, Jae H Choi, Steven P Gygi, Qing Yu, Joao A Paulo.

  Targeted proteomics strategies present a streamlined hypothesis-driven approach to analyze specific sets of pathways or disease related proteins. goDig is a quantitative, targeted tandem mass tag (TMT)-based assay that can measure the relative abundance differences for hundreds of proteins directly from unfractionated mixtures. Specific protein groups or entire pathways of up to 200 proteins can be selected for quantitative profiling, while leveraging sample multiplexing permits the simultaneous analysis of up to 18 samples. Despite these benefits, implementing goDig is not without challenges, as it requires access to an instrument application programming interface (iAPI), an elution order and spectral library, a web-based method builder, and dedicated companion software. In addition, the absence of an example test assay may dissuade researchers from testing or implementing goDig. Here, we repurpose the TKO11 standard─which is commercially available but may also be assembled in-lab─and establish it as a de facto test assay for goDig. We build a proteome-wide goDig yeast library, quantify protein expression across several gene ontology (GO) categories, and compare these results to a fully fractionated yeast gold-standard data set. Essentially, we provide a guide detailing the goDig-based quantification of TKO11, which can also be used as a template for user-defined assays in other species.

Keywords:  API; GoDig; TKO; TMT; TMTpro; targeted assay

DOI:  https://doi.org/10.1021/acs.jproteome.3c00493
Methods Mol Biol. 2024 ;2737 377-386

Quantitative LC-MS/MS Analysis of Opiates and Opioids in Human Urine.

Putuma P Gqamana, Y Victoria Zhang.

  We hereby present a direct, clinical LC-MS/MS assay for the simultaneous analysis of opiates and opioids in human urine. The assay is used as confirmations as well as a quantitative assay following immunoassay urine drug screens. Hence, the utility is extended to report opiate/opioid levels in the range 10-1000 ng/mL for each of the 13 analytes, which include morphine and metabolites; codeine and metabolites; as well as synthetics such as heroin metabolites, dihydrocodeine, oxycodone, and naloxone. The assay employs dilute and shoot, utilizing reversed-phase liquid chromatography for mixture separation and positive-mode electrospray-ionization multiple-reaction-monitoring for MS detection in the TSQ Endura triple-quadrupole (QqQ) instrument. Thereafter, quantitative analysis is based on the linear calibration using six reference standards, whereby the instrument response for each analyte at a given concentration is normalized against stable isotope labeled internal standard.

Keywords:  Codeine; Heroin metabolites; LC-MS/MS; Metabolites; Morphine; Multiple-reaction-monitoring (MRM); Opiates; Synthetic opioids

DOI:  https://doi.org/10.1007/978-1-0716-3541-4_34
Cancer Res. 2023 Dec 01.

Adipose triglyceride lipase is a therapeutic target in advanced prostate cancer that promotes metabolic plasticity.

Dominik Awad, Pham Hong Anh Cao, Thomas L Pulliam, Meredith Spradlin, Elavarasan Subramani, Tristen V Tellman, Caroline F Ribeiro, Riccardo Muzzioli, Brittany E Jewell, Hubert Pakula, Jeffrey J Ackroyd, Mollianne M Murray, Jenny J Han, Mei Leng, Antrix Jain, Badrajee Piyarathna, JIngjing Liu, Xingzhi Song, Jianhua Zhang, Albert R Klekers, Justin M Drake, Michael M Ittmann, Cristian Coarfa, David Piwnica-Worms, Mary C Farach-Carson, Massimo Loda, Livia S Eberlin, Daniel E Frigo.

Lipid metabolism plays a central role in prostate cancer. To date, the major focus has centered on de novo lipogenesis and lipid uptake in prostate cancer, but inhibitors of these processes have not benefited patients. Better understanding of how cancer cells access lipids once they are created or taken up and stored could uncover more effective strategies to perturb lipid metabolism and treat patients. Here, we identified that expression of adipose triglyceride lipase (ATGL), an enzyme that controls lipid droplet homeostasis and a previously suspected tumor suppressor, correlates with worse overall survival in men with advanced, castration-resistant prostate cancer (CRPC). Molecular, genetic, or pharmacological inhibition of ATGL impaired human and murine prostate cancer growth in vivo and in cell culture or organoids under conditions mimicking the tumor microenvironment. Mass spectrometry imaging demonstrated ATGL profoundly regulates lipid metabolism in vivo, remodeling membrane composition. ATGL inhibition induced metabolic plasticity, causing a glycolytic shift that could be exploited therapeutically by co-targeting both metabolic pathways. Patient-derived phosphoproteomics identified ATGL serine 404 as a target of CAMKK2-AMPK signaling in CRPC cells. Mutation of serine 404 did not alter the lipolytic activity of ATGL but did decrease CRPC growth, migration, and invasion, indicating that non-canonical ATGL activity also contributes to disease progression. Unbiased immunoprecipitation/mass spectrometry suggested that mutation of serine 404 not only disrupts existing ATGL protein interactions but also leads to new protein-protein interactions. Together, these data nominate ATGL as a therapeutic target for CRPC and provide insights for future drug development and combination therapies.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-0555
Nat Immunol. 2023 Dec;24(12): 2008-2020

The immunometabolic ecosystem in cancer.

Glenn R Bantug, Christoph Hess.

Our increased understanding of how key metabolic pathways are activated and regulated in malignant cells has identified metabolic vulnerabilities of cancers. Translating this insight to the clinics, however, has proved challenging. Roadblocks limiting efficacy of drugs targeting cancer metabolism may lie in the nature of the metabolic ecosystem of tumors. The exchange of metabolites and growth factors between cancer cells and nonmalignant tumor-resident cells is essential for tumor growth and evolution, as well as the development of an immunosuppressive microenvironment. In this Review, we will examine the metabolic interplay between tumor-resident cells and how targeted inhibition of specific metabolic enzymes in malignant cells could elicit pro-tumorigenic effects in non-transformed tumor-resident cells and inhibit the function of tumor-specific T cells. To improve the efficacy of metabolism-targeted anticancer strategies, a holistic approach that considers the effect of metabolic inhibitors on major tumor-resident cell populations is needed.

DOI: https://doi.org/10.1038/s41590-023-01675-y
Methods Mol Biol. 2024 ;2737 443-452

Quantification of Thiopurine Metabolites in Human Erythrocytes by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Lie Li, Natalya Atkinson, Kristine R Crews, Alejandro R Molinelli.

  The thiopurine drugs, azathioprine, mercaptopurine, and thioguanine, are widely used in the treatment of several malignant and nonmalignant diseases. These inactive prodrugs undergo extensive metabolism to form active cytotoxic metabolites, which act mainly by incorporating into DNA and affecting cell replication. Thiopurine methyltransferase is a highly variable cytosolic enzyme that catalyzes the S-methylation of the thiopurine bases-an inactivating pathway. Patients with low-activity variants of TPMT can be affected by pronounced pharmacologic effects when receiving thiopurine medications. Clinical studies have reported significant interpatient variability in intracellular thiopurine metabolite concentrations in patients receiving thiopurine therapy. In this chapter, we present an LC-MS/MS method to monitor the thiopurine metabolites: 6-thioguanine nucleotides and 6-methylmercaptopurine derivatives in human erythrocytes. This method utilizes acid hydrolysis to release the bases and improves upon previously published procedures by utilizing stable isotope internal standards and a more efficient chromatographic separation.

Keywords:  LC-MS/MS; Mercaptopurine; Therapeutic drug monitoring; Thioguanine

DOI:  https://doi.org/10.1007/978-1-0716-3541-4_41
Methods Mol Biol. 2024 ;2737 79-90

Analysis of Barbiturates in Urine by LC-MS/MS.

Matthew Campbell, Gregory Janis, Hunter Horne, Hemamalini Ketha.

  In the method described here, an aliquot of a urine sample is analyzed to detect barbiturates through dilution and ultra-high-performance chromatography-tandem mass spectrometry (UPLC-MS/MS) using deuterated internal standards. This assay detects the presence of nine barbiturate drugs-amobarbital, barbital, butalbital, butabarbital, mephobarbital, secobarbital, pentobarbital, phenobarbital, and thiopental. This protocol describes two LC separation methods-first LC method (2.2 min/sample) is intended to be used as a first step of the analysis that does not separate amobarbital and pentobarbital, and a second, longer (2.7 min/sample) LC method is intended to be used only for samples which have a peak in the amobarbital/pentobarbital retention time on the shorter LC method. Since the frequency at which amobarbital and pentobarbital are observed in clinical populations is low, the shorter LC method helps gain efficiency in a high-volume laboratory environment. Additional features of this protocol that help in efficiency gain are automated extraction using Hamilton™ liquid handling system and algorithmic data review using Ascent™ software.

Keywords:  Barbiturates; Distinguishing amobarbital and pentobarbital; Liquid chromatography; Mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-3541-4_8
Methods Mol Biol. 2024 ;2737 347-357

Quantification of Olanzapine by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Kamisha L Johnson-Davis, Diane Ly.

  Olanzapine (Zyprexa™) is indicated for the treatment of schizophrenia and bipolar disorder. Olanzapine is metabolized to inactive metabolites; therefore, this assay measures the concentrations of olanzapine and internal standard, olanzapine-D3. This procedure provides instructions for quantitating levels of olanzapine in blood serum specimens. An internal standard (IS) solution consisting of the deuterated analogue of olanzapine is added to an aliquot of the patient serum or plasma, standards, and positive controls. The drugs of interest are separated from the biological fluid using solid phase extraction. The dried extracts are reconstituted in mobile phase and separated by liquid chromatography. The solution is introduced into an Agilent 6470A triple quadrupole tandem mass spectrometer via positive mode electrospray ionization (ESI) utilizing multiple reaction monitoring (MRM). Quantitation is performed by comparison to a calibration curve, using Indigo® (Ascent) software.

Keywords:  Mass spectrometry; Olanzapine; Plasma; Serum

DOI:  https://doi.org/10.1007/978-1-0716-3541-4_32
Bio Protoc. 2023 Nov 20. 13(22): e4880

A New, Rapid Method for the Quantification of Dolichyl Phosphates in Cell Cultures Using TMSD Methylation Combined with LC-MS Analysis.

Dipali Kale, Timo Sachsenheimer, Albert Sickmann, Britta Brügger.

  Dolichyl phosphates (DolP) are ubiquitous lipids that are present in almost all eukaryotic membranes. They play a key role in several protein glycosylation pathways and the formation of glycosylphosphatidylinositol anchors. These lipids constitute only ~0.1% of total phospholipids, and their analysis by reverse phase (RP) liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is challenging due to their high lipophilicity (log P > 20), poor ionization efficiency, and relatively low abundance. To overcome these challenges, we have introduced a new approach for DolP analysis by combining trimethylsilyldiazomethane (TMSD)-based phosphate methylation and HRMS analysis. The analytical method was validated for its reproducibility, sensitivity, and accuracy. The established workflow was successfully applied for the simultaneous characterization and quantification of DolP species with different isoprene units in lipid extracts of HeLa and Saccharomyces cerevisiae cells.

Keywords:  DolP; Dolichyl phosphate; LC-MS; LC–HRMS; Lipid analysis; Methylation; TMSD

DOI:  https://doi.org/10.21769/BioProtoc.4880
Front Mol Biosci. 2023 ;10 1283083

A one-year pilot study comparing direct-infusion high resolution mass spectrometry based untargeted metabolomics to targeted diagnostic screening for inherited metabolic diseases.

Anke P Willems, Maria van der Ham, Birgit G M Schiebergen-Bronkhorst, Mirjam van Aalderen, Martina M J de Barse, Fini E De Gruyter, Ilja N van Hoek, Mia L Pras-Raves, Monique G M de Sain-van der Velden, Hubertus C M T Prinsen, Nanda M Verhoeven-Duif, Judith J M Jans.

  Background: Early diagnosis of inherited metabolic diseases (IMDs) is important because treatment may lead to reduced mortality and improved prognosis. Due to their diversity, it is a challenge to diagnose IMDs in time, effecting an emerging need for a comprehensive test to acquire an overview of metabolite status. Untargeted metabolomics has proven its clinical potential in diagnosing IMDs, but is not yet widely used in genetic metabolic laboratories. Methods: We assessed the potential role of plasma untargeted metabolomics in a clinical diagnostic setting by using direct infusion high resolution mass spectrometry (DI-HRMS) in parallel with traditional targeted metabolite assays. We compared quantitative data and qualitative performance of targeted versus untargeted metabolomics in patients suspected of an IMD (n = 793 samples) referred to our laboratory for 1 year. To compare results of both approaches, the untargeted data was limited to polar metabolites that were analyzed in targeted plasma assays. These include amino acid, (acyl)carnitine and creatine metabolites and are suitable for diagnosing IMDs across many of the disease groups described in the international classification of inherited metabolic disorders (ICIMD). Results: For the majority of metabolites, the concentrations as measured in targeted assays correlated strongly with the semi quantitative Z-scores determined with DI-HRMS. For 64/793 patients, targeted assays showed an abnormal metabolite profile possibly indicative of an IMD. In 55 of these patients, similar aberrations were found with DI-HRMS. The remaining 9 patients showed only marginally increased or decreased metabolite concentrations that, in retrospect, were most likely to be clinically irrelevant. Illustrating its potential, DI-HRMS detected additional patients with aberrant metabolites that were indicative of an IMD not detected by targeted plasma analysis, such as purine and pyrimidine disorders and a carnitine synthesis disorder. Conclusion: This one-year pilot study showed that DI-HRMS untargeted metabolomics can be used as a first-tier approach replacing targeted assays of amino acid, acylcarnitine and creatine metabolites with ample opportunities to expand. Using DI-HRMS untargeted metabolomics as a first-tier will open up possibilities to look for new biomarkers.

Keywords:  biomarker; diagnostics; direct-infusion high resolution mass spectrometry; genetic diseases; inherited metabolic diseases; untargeted metabolomics

DOI:  https://doi.org/10.3389/fmolb.2023.1283083
Methods Mol Biol. 2024 ;2737 1-13

Mass Spectrometry in Clinical Laboratory: Applications in Therapeutic Drug Monitoring and Toxicology.

Y Victoria Zhang, Uttam Garg.

  Mass spectrometry (MS) coupled with liquid chromatography (LC) or gas chromatography (GC) has been proven to be a powerful platform in research and specialized clinical laboratories for decades. In clinical laboratories, it is used for compound identification and quantification. Due to the ability to provide specific identification, high sensitivity, and simultaneous analysis of multiple analytes (>100) in recent years, application of MS in routine clinical laboratories has increased significantly. Although MS is used in many laboratory areas, therapeutic drug monitoring, drugs of abuse, and clinical toxicology remain the primary focuses of the field. Due to rapid increase in the number of prescription drugs and drugs of abuse (e.g., novel psychoactive substances), clinical laboratories are challenged with developing new MS assays to meet the clinical needs of the patients. We are here to present "off-the-shelf" and "ready-to-use" protocols of recent developments in new assays to help the clinical laboratory community adopt the technology and analysis for the betterment of patient care.

Keywords:  Clinical laboratory; Drugs; Gas chromatography; Immunoassays; Liquid chromatography; Mass spectrometry; Tandem mass spectrometry; Therapeutic drug monitoring; Time of flight; Toxicology

DOI:  https://doi.org/10.1007/978-1-0716-3541-4_1
Anal Chem. 2023 Nov 30.

CHRISTMAS: Chiral Pair Isobaric Labeling Strategy for Multiplexed Absolute Quantitation of Enantiomeric Amino Acids.

Zhijun Zhu, Shuling Xu, Zicong Wang, Daniel G Delafield, Michael J Rigby, Gaoyuan Lu, Ting-Jia Gu, Peng-Kai Liu, Min Ma, Luigi Puglielli, Lingjun Li.

Amino acids (AAs) in the d-form are involved in multiple pivotal neurological processes, although their l-enantiomers are most commonly found. Mass spectrometry-based analysis of low-abundance d-AAs has been hindered by challenging enantiomeric separation from l-AAs, low sensitivity for detection, and lack of suitable internal standards for accurate quantification. To address these critical gaps, N,N-dimethyl-l-leucine (l-DiLeu) tags are first validated as novel chiral derivatization reagents for chromatographic separation of 20 pairs of d/l-AAs, allowing the construction of a 4-plex isobaric labeling strategy for enantiomer-resolved quantification through single step tagging. Additionally, the creative design of N,N-dimethyl-d-leucine (d-DiLeu) reagents offers an alternative approach to generate analytically equivalent internal references of d-AAs using d-DiLeu-labeled l-AAs. By labeling cost-effective l-AA standards using paired d- and l-DiLeu, this approach not only enables absolute quantitation of both d-AAs and l-AAs from complex biological matrices with enhanced precision but also significantly boosts the combined signal intensities from all isobaric channels, greatly improving the detection and quantitation of low-abundance AAs, particularly d-AAs. We term this quantitative strategy CHRISTMAS, which stands for chiral pair isobaric labeling strategy for multiplexed absolute quantitation. Leveraging the ion mobility collision cross section (CCS) alignment, interferences from coeluting isomers/isobars are effectively filtered out to provide improved quantitative accuracy. From wild-type and Alzheimer's disease (AD) mouse brains, we successfully quantified 20 l-AAs and 5 d-AAs. The significant presence and differential trends of certain d-AAs compared to those of their l-counterparts provide valuable insights into the involvement of d-AAs in aging, AD progression, and neurodegeneration.

DOI: https://doi.org/10.1021/acs.analchem.3c03847
Methods Mol Biol. 2024 ;2737 123-132

Quantitative Analysis of Buprenorphine, Norbuprenorphine, and Their Glucuronide Metabolites in Human Urine.

Putuma P Gqamana, Daniel Rudy, Y Victoria Zhang.

  We present a quantitative clinical LC-MS/MS assay for the simultaneous analysis of buprenorphine, norbuprenorphine, and their glucuronide metabolites in human urine. The assay is based on the direct and hydrolysis-free sample preparation protocol, i.e., dilute and shoot, whereby clarified urine specimens are diluted in internal standard reagent and injected into the LC-MS/MS instrument. The analytical platform employs reversed-phase liquid chromatography for upfront separation and electrospray ionization multiple reaction monitoring MS detection via the triple-quadrupole (TSQ Quantiva) instrument. The assay has a quantitative analytical range of 5 ng/mL-1000 ng/mL represented at seven levels for each of the four analytes. A unique stable isotopically labeled analogue is used as internal standard for each analyte. For high-throughput performance, the assay can be multiplexed between two LC channels.

Keywords:  Buprenorphines; Electrospray ionization (ESI); Glucuronides; High-throughput LC-MS/MS; Multiple reaction monitoring (MRM); Therapeutic drug monitoring

DOI:  https://doi.org/10.1007/978-1-0716-3541-4_12
Adv Exp Med Biol. 2024 ;1440 57-71

LC-MS Approaches for Oxysterols in Various Biosamples.

Kristina S Kømurcu, Steven R Wilson, Hanne Røberg-Larsen.

  Oxysterols are involved in a plethora of biological processes, including a wide variety of diseases. Therefore, monitoring oxysterols is important for obtaining a deeper understanding of their biological roles and utilizing them as, for example, biomarkers. However, oxysterols can be challenging compounds to study, as they can be very similar in chemical structure but still have distinct biological roles. In addition, oxysterols may be difficult to detect, even with advanced analytical instrumentation. We here focus on the use of liquid chromatography-mass spectrometry (LC-MS) for the analysis of oxysterols, with an additional focus on the steps needed to prepare oxysterols for LC-MS. Steps can include chemical modification of the oxysterols for improving LC-MS sensitivity and adding chemicals that can reveal if the oxysterol levels have been perturbed during preparation. We then round off with descriptions and applications of various sample preparations for different biological matrices, from blood to cells, and biosamples with emerging attention, for example, exosomes and organoids. Taken together, oxysterol analysis is highly compatible with a wide variety of biosamples, allowing for a deeper understanding of these challenging analytes.

Keywords:  Biosamples; Derivatization; Exosome; Liquid chromatography–mass spectrometry; Organoids; Oxysterols; Sample preparation

DOI:  https://doi.org/10.1007/978-3-031-43883-7_4
bioRxiv. 2023 Nov 14. pii: 2023.11.10.566378. [Epub ahead of print]

SpatialCells: Automated Profiling of Tumor Microenvironments with Spatially Resolved Multiplexed Single-Cell Data.

Guihong Wan, Zoltan Maliga, Boshen Yan, Tuulia Vallius, Yingxiao Shi, Sara Khattab, Crystal Chang, Ajit J Nirmal, Kun-Hsing Yu, David Liu, Christine G Lian, Mia S DeSimone, Peter K Sorger, Yevgeniy R Semenov.

Background: Cancer is a complex cellular ecosystem where malignant cells coexist and interact with immune, stromal, and other cells within the tumor microenvironment. Recent technological advancements in spatially resolved multiplexed imaging at single-cell resolution have led to the generation of large-scale and high-dimensional datasets from biological specimens. This underscores the necessity for automated methodologies that can effectively characterize the molecular, cellular, and spatial properties of tumor microenvironments for various malignancies.
Results: This study introduces SpatialCells, an open-source software package designed for region-based exploratory analysis and comprehensive characterization of tumor microenvironments using multiplexed single-cell data.
Conclusions: SpatialCells efficiently streamlines the automated extraction of features from multiplexed single-cell data and can process samples containing millions of cells. Thus, SpatialCells facilitates subsequent association analyses and machine learning predictions, making it an essential tool in advancing our understanding of tumor growth, invasion, and metastasis.
Availability of code and materials: https://github.com/SemenovLab/SpatialCells .

DOI: https://doi.org/10.1101/2023.11.10.566378
Front Neurol. 2023 ;14 1292160

Novel approaches targeting ferroptosis in treatment of glioma.

Jing Zhao, Fengling Zang, Xiaoya Huo, Shengzhe Zheng.

  Glioma is a malignant brain tumor with a high mortality rate; hence novel treatment approaches are being explored to improve patient outcomes. Ferroptosis, a newly described form of regulated cell death, is emerging as a potential therapeutic target in glioma. Ferroptosis is characterized by the accumulation of lipid peroxides due to a loss of intracellular antioxidant systems represented by the depletion of glutathione and decreased activity of glutathione peroxidase 4 (GPX4). Since glioma cells have a high demand for iron and lipid metabolism, modulation of ferroptosis may represent a promising therapeutic approach for this malignancy. Recent studies indicate that ferroptosis inducers like erastin and RSL3 display potent anticancer activity in a glioma model. In addition, therapeutic strategies, including GPX4 targeting, lipid metabolism modulation, inhibition of amino acid transporters, and ferroptosis targeting natural compounds, have shown positive results in preclinical studies. This review will provide an overview of the functions of ferroptosis in glioma and its potential as a suitable target for glioma therapy.

Keywords:  cancer; erastin; glutathione peroxidase 4; natural compounds; reactive oxygen species

DOI:  https://doi.org/10.3389/fneur.2023.1292160
Anal Methods. 2023 Nov 27.

Simultaneous separation and identification of all structural isomers and enantiomers of aminobutyric acid using a highly sensitive chiral resolution labeling reagent.

Makoto Ozaki, Motoshi Shimotsuma, Takefumi Kuranaga, Hideaki Kakeya, Tsunehisa Hirose.

Aminobutyric acid has structural isomers (α-, β-, and γ-aminobutyric acids) and enantiomers (D/L-forms) with various unique functions. Therefore, a quantitative method for determining the content of each aminobutyric acid must be developed. In general, quantitative simultaneous analysis of multiple compounds is conducted via high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). However, simultaneous separation and highly sensitive detection of all aminobutyric acids are complicated, so highly sensitive analytical methods for the separation and identification of each compound have not yet been established. We previously developed highly sensitive chiral resolution labeling reagents. Herein, we propose a highly sensitive analytical method for the simultaneous separation and identification of all aminobutyric acids via LC-MS and labeling with our original highly sensitive chiral resolution labeling reagent, 1-fluoro-2,4-dinitrophenyl-5-L-valine-N,N-dimethylethylenediamine amide (L-FDVDA). The labeling reagent was completely bound to all aminobutyric acids through incubation overnight (>15 h) at 50 °C. Additionally, the labeled aminobutyric acids could be stored for at least 1 week at 4 °C. Furthermore, we demonstrated simultaneous separation and identification of aminobutyric acids in biological samples and foods through LC-MS using a C18 column after labeling with L-FDVDA. Our method is expected to be adopted for the analysis of the contents of all aminobutyric acids in biological and clinical samples as well as various foods.

DOI: https://doi.org/10.1039/d3ay01665j
MedComm (2020). 2023 Dec;4(6): e434

Tumor-derived extracellular vesicles and particles (EVPs): pivotal vectors in driving metabolic reprogramming.

Xinming Su, Peijie Zheng, Shiwei Duan.



Keywords:  cancer; chemotherapy toxicity; extracellular vesicles and particles; liver metabolism; metabolic reprogramming; nonalcoholic fatty liver disease

DOI:  https://doi.org/10.1002/mco2.434
Mol Ther Oncolytics. 2023 Dec 19. 31 100740

Targeting the lactic acid metabolic pathway for antitumor therapy.

Zhi Li, Jiuwei Cui.

  Lactic acid is one of the most abundant products of cellular metabolism and has historically been considered a cell-damaging metabolic product. However, as research has deepened, the beneficial effects of lactic acid on tumor cells and the tumor microenvironment have received increasing attention from the oncology community. Lactic acid can not only provide tumor cells with energy but also act as a messenger molecule that promotes tumor growth and progression and protects tumor cells from immune cells and killing by radiation and chemotherapy. Thus, the inhibition of tumor cell lactic acid metabolism has emerged as a novel antitumor treatment strategy that can also effectively enhance the efficacy of conventional antitumor therapies. In this review, we classify the currently available therapies targeting lactic acid metabolism and examine their prospects for clinical application.

Keywords:  acidic tumor microenvironment; cancer; drug delivery; lactic acid metabolism; targeted therapy

DOI:  https://doi.org/10.1016/j.omto.2023.100740
ArXiv. 2023 Nov 13. pii: arXiv:2311.07791v1. [Epub ahead of print]

Comprehensive Overview of Bottom-up Proteomics using Mass Spectrometry.

Yuming Jiang, Devasahayam Arokia Balaya Rex, Dina Schuster, Benjamin A Neely, Germán L Rosano, Norbert Volkmar, Amanda Momenzadeh, Trenton M Peters-Clarke, Susan B Egbert, Simion Kreimer, Emma H Doud, Oliver M Crook, Amit Kumar Yadav, Muralidharan Vanuopadath, Martín L Mayta, Anna G Duboff, Nicholas M Riley, Robert L Moritz, Jesse G Meyer.

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics.
Methods Mol Biol. 2024 ;2747 55-66

Expression and Purification of Recombinant ADAMTS8.

Tina Burkhard, Alexander Frederick Minns, Salvatore Santamaria.

  ADAMTS8 (A Disintegrin-like and Metalloproteinase with Thrombospondin motifs 8) is a secreted zinc-dependent metalloproteinase whose expression is downregulated in a variety of solid tumors. Xenografts expressing high levels of ADAMTS8 have a poor capacity to invade and migrate in nude mice. While this data highlights a beneficial, anti-cancerogenic role of ADAMTS8, the mechanism behind this activity is still not fully elucidated. So far, the only reported substrate for ADAMTS8 is osteopontin (OPN), an extracellular matrix protein widely implicated in multiple steps of cancer progression, albeit, similar to other ADAMTS family members, it is very likely that ADAMTS8 cleaves a variety of substrates. The availability of purified ADAMTS8 may enlighten the biological role of this metalloproteinase.Here we describe methods for expression and purification of recombinant ADAMTS8 in HEK293T cells as well as a convenient assay to test ADAMTS8 proteolytic activity using OPN as a substrate.

Keywords:  ADAMTS8; Extracellular matrix; Osteopontin

DOI:  https://doi.org/10.1007/978-1-0716-3589-6_5