bims-mascan 2022-02-20 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2022–02–20
25 papers selected by
Giovanny Rodríguez Blanco, University of Edinburgh

Stable Isotope Tracing and Metabolomics to Study In Vivo Brown Adipose Tissue Metabolic Fluxes.
Low Energy Status under Methionine Restriction Is Essentially Independent of Proliferation or Cell Contact Inhibition.
Signaling Lipidomic Analysis of Thermogenic Adipocytes.
Expression of 3-Methylcrotonyl-CoA Carboxylase in Brain Tumors and Capability to Catabolize Leucine by Human Neural Cancer Cells.
Advancing Cancer Treatment by Targeting Glutamine Metabolism-A Roadmap.
CPExtract, a Software Tool for the Automated Tracer-Based Pathway Specific Screening of Secondary Metabolites in LC-HRMS Data.
An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids.
Dietary PUFA drives diverse systems-level changes in lipid metabolism.
Interactions between Radiation and One-Carbon Metabolism.
Reproducible untargeted metabolomics workflow for exhaustive MS2 data acquisition of MS1 features.
Differential Proteomic Analysis of Complex Mixtures by Label-Free nLC MS/MS.
Automated Annotation of Untargeted All-Ion Fragmentation LC-MS Metabolomics Data with MetaboAnnotatoR.
De novo serine synthesis regulates chondrocyte proliferation during bone development and repair.
Proteomic Analysis of Whole Blood Using Volumetric Absorptive Microsampling for Precision Medicine Biomarker Studies.
Changing Metabolic Patterns along the Colorectal Adenoma-Carcinoma Sequence.
Metabolic modulation of immune checkpoints and novel therapeutic strategies in cancer.
The Application of Metabolomics in Recent Colorectal Cancer Studies: A State-of-the-Art Review.
Ketone body oxidation increases cardiac endothelial cell proliferation.
Evaluation of endogenous peptide stereochemistry using liquid chromatography-mass spectrometry-based spiking experiments.
Untargeted detection of the carbonyl metabolome by chemical derivatization and liquid chromatography-tandem mass spectrometry in precursor ion scan mode: Elucidation of COVID-19 severity biomarkers.
Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity.
Equivalent Carbon Number and Interclass Retention Time Conversion Enhance Lipid Identification in Untargeted Clinical Lipidomics.
Fatty acid transport protein 2 interacts with ceramide synthase 2 to promote ceramide synthesis.
Model of retention time and density of gradient peak capacity for improved LC-MS method optimization: Application to metabolomics.
Accurate determination of amino acids by quadruple isotope dilution-reverse phase liquid Chromatography-Tandem mass spectrometry after derivatization with 2-Naphthoyl chloride.

Methods Mol Biol. 2022 ;2448 119-130

Stable Isotope Tracing and Metabolomics to Study In Vivo Brown Adipose Tissue Metabolic Fluxes.

Su Myung Jung, Johnny Le, Will G Doxsey, John A Haley, Grace Park, David A Guertin, Cholsoon Jang.

  Brown adipose tissue (BAT) demonstrates extraordinary metabolic capacity. Previous research using conventional radio tracers reveals that BAT can act as a sink for a diverse menu of nutrients; still, the question of how BAT utilizes these nutrients remains unclear. Recent advances in mass spectrometry (MS) coupled to stable isotope tracing methods have greatly improved our understanding of metabolism in biology. Here, we have developed a BAT-tailored metabolomics and stable isotope tracing protocol using, as an example, the universally labeled 13C-glucose, a key nutrient heavily utilized by BAT. This method enables metabolic roadmaps to be drawn and pathway fluxes to be inferred for each nutrient tracer within BAT and its application could uncover new metabolic pathways not previously appreciated for BAT physiology.

Keywords:  Brown adipose tissue; Brown fat; Gavage; Glucose; Liquid chromatography-mass spectrometry (LC-MS); Metabolism; Metabolomics; Stable isotope tracing; Temperature acclimation

DOI:  https://doi.org/10.1007/978-1-0716-2087-8_8
Cells. 2022 Feb 04. pii: 551. [Epub ahead of print]11(3):

Low Energy Status under Methionine Restriction Is Essentially Independent of Proliferation or Cell Contact Inhibition.

Corinna Koderer, Werner Schmitz, Anna Chiara Wünsch, Julia Balint, Mohamed El-Mesery, Julian Manuel Volland, Stefan Hartmann, Christian Linz, Alexander Christian Kübler, Axel Seher.

  Nonlimited proliferation is one of the most striking features of neoplastic cells. The basis of cell division is the sufficient presence of mass (amino acids) and energy (ATP and NADH). A sophisticated intracellular network permanently measures the mass and energy levels. Thus, in vivo restrictions in the form of amino acid, protein, or caloric restrictions strongly affect absolute lifespan and age-associated diseases such as cancer. The induction of permanent low energy metabolism (LEM) is essential in this process. The murine cell line L929 responds to methionine restriction (MetR) for a short time period with LEM at the metabolic level defined by a characteristic fingerprint consisting of the molecules acetoacetate, creatine, spermidine, GSSG, UDP-glucose, pantothenate, and ATP. Here, we used mass spectrometry (LC/MS) to investigate the influence of proliferation and contact inhibition on the energy status of cells. Interestingly, the energy status was essentially independent of proliferation or contact inhibition. LC/MS analyses showed that in full medium, the cells maintain active and energetic metabolism for optional proliferation. In contrast, MetR induced LEM independently of proliferation or contact inhibition. These results are important for cell behaviour under MetR and for the optional application of restrictions in cancer therapy.

Keywords:  L929; LC/MS; amino acid; caloric restriction; contact inhibition; liquid chromatography/mass spectrometry; mass spectrometry; metabolomics; methionine restriction; proliferation

DOI:  https://doi.org/10.3390/cells11030551
Methods Mol Biol. 2022 ;2448 251-271

Signaling Lipidomic Analysis of Thermogenic Adipocytes.

Sean D Kodani, Valerie Bussberg, Niven R Narain, Michael A Kiebish, Yu-Hua Tseng.

  Brown adipose tissue is a thermogenic organ that possesses anti-diabetic and anti-obesogenic potential. There has recently been growing interest on the secretory role of brown adipose tissue in regulating whole-body metabolism. Several signaling lipids, including 12-HEPE and 12,13-diHOME, have been shown to be secreted by brown adipose tissue and have demonstrated roles in regulating whole-body energy metabolism. Lipidomics platforms that broadly characterize the signaling lipidome can deconvolute the underlying biology of the lipid metabolites having a broad systemic impact on physiology. Herein, we describe how to perform and analyze LC-MS/MS signaling lipidomics on mature brown adipocytes.

Keywords:  Brown adipocyte; LC-MS/MS; Lipidomic analysis; Lipoxygenase; Signaling lipids

DOI:  https://doi.org/10.1007/978-1-0716-2087-8_16
Cancers (Basel). 2022 Jan 24. pii: 585. [Epub ahead of print]14(3):

Expression of 3-Methylcrotonyl-CoA Carboxylase in Brain Tumors and Capability to Catabolize Leucine by Human Neural Cancer Cells.

Eduard Gondáš, Alžbeta Kráľová Trančíková, Eva Baranovičová, Jakub Šofranko, Jozef Hatok, Bhavani S Kowtharapu, Tomáš Galanda, Dušan Dobrota, Peter Kubatka, Dietrich Busselberg, Radovan Murín.

  Leucine is an essential, ketogenic amino acid with proteinogenic, metabolic, and signaling roles. It is readily imported from the bloodstream into the brain parenchyma. Therefore, it could serve as a putative substrate that is complementing glucose for sustaining the metabolic needs of brain tumor cells. Here, we investigated the ability of cultured human cancer cells to metabolize leucine. Indeed, cancer cells dispose of leucine from their environment and enrich their media with the metabolite 2-oxoisocaproate. The enrichment of the culture media with a high level of leucine stimulated the production of 3-hydroxybutyrate. When 13C6-leucine was offered, it led to an increased appearance of the heavier citrate isotope with a molar mass greater by two units in the culture media. The expression of 3-methylcrotonyl-CoA carboxylase (MCC), an enzyme characteristic for the irreversible part of the leucine catabolic pathway, was detected in cultured cancer cells and human tumor samples by immunoprobing methods. Our results demonstrate that these cancer cells can catabolize leucine and furnish its carbon atoms into the tricarboxylic acid (TCA) cycle. Furthermore, the release of 3-hydroxybutyrate and citrate by cancer cells suggests their capability to exchange these metabolites with their milieu and the capability to participate in their metabolism. This indicates that leucine could be an additional substrate for cancer cell metabolism in the brain parenchyma. In this way, leucine could potentially contribute to the synthesis of metabolites such as lipids, which require the withdrawal of citrate from the TCA cycle.

Keywords:  3-methylcrotonyl-CoA carboxylase; acetyl-CoA; branched-chain amino acid; cancer cells; citrate; ketone bodies; leucine; metabolism

DOI:  https://doi.org/10.3390/cancers14030585
Cancers (Basel). 2022 Jan 22. pii: 553. [Epub ahead of print]14(3):

Advancing Cancer Treatment by Targeting Glutamine Metabolism-A Roadmap.

Anna Halama, Karsten Suhre.

  Tumor growth and metastasis strongly depend on adapted cell metabolism. Cancer cells adjust their metabolic program to their specific energy needs and in response to an often challenging tumor microenvironment. Glutamine metabolism is one of the metabolic pathways that can be successfully targeted in cancer treatment. The dependence of many hematological and solid tumors on glutamine is associated with mitochondrial glutaminase (GLS) activity that enables channeling of glutamine into the tricarboxylic acid (TCA) cycle, generation of ATP and NADPH, and regulation of glutathione homeostasis and reactive oxygen species (ROS). Small molecules that target glutamine metabolism through inhibition of GLS therefore simultaneously limit energy availability and increase oxidative stress. However, some cancers can reprogram their metabolism to evade this metabolic trap. Therefore, the effectiveness of treatment strategies that rely solely on glutamine inhibition is limited. In this review, we discuss the metabolic and molecular pathways that are linked to dysregulated glutamine metabolism in multiple cancer types. We further summarize and review current clinical trials of glutaminolysis inhibition in cancer patients. Finally, we put into perspective strategies that deploy a combined treatment targeting glutamine metabolism along with other molecular or metabolic pathways and discuss their potential for clinical applications.

Keywords:  cancer; cancer treatment; drug resistance; glutamine metabolism; glutaminolysis inhibition; metabolism

DOI:  https://doi.org/10.3390/cancers14030553
Anal Chem. 2022 Feb 15.

CPExtract, a Software Tool for the Automated Tracer-Based Pathway Specific Screening of Secondary Metabolites in LC-HRMS Data.

Bernhard Seidl, Rainer Schuhmacher, Christoph Bueschl.

The use of stable isotopically labeled tracers is a long-proven way of specifically detecting and tracking derived metabolites through a metabolic network of interest. While the recently developed stable isotope-assisted methods and associated, supporting data analysis tools have greatly improved untargeted metabolomics approaches, no software tool is currently available that allows us to automatically and flexibly search liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) chromatograms for user-definable isotopolog patterns expected for the metabolism of labeled tracer substances. Here, we present Custom Pattern Extract (CPExtract), a versatile software tool that allows for the first time the high-throughput search for user-defined isotopolog patterns in LC-HRMS data. The patterns can be specified via a set of rules including the presence or absence of certain isotopologs, their relative intensity ratios as well as chromatographic coelution. Each isotopolog pattern satisfying the respective rules is verified on an MS scan level and also in the chromatographic domain. The CPExtract algorithm allows the use of both labeled tracer compounds in nonlabeled biological samples as well as a reversed tracer approach, employing nonlabeled tracer compounds along with globally labeled biological samples. In a proof-of-concept study, we searched for metabolites specifically arising from the malonate pathway of the filamentous fungi Fusarium graminearum and Trichoderma reesei. 1,2,3-13C3-malonic acid diethyl ester and native malonic acid monomethyl ester were used as tracers. We were able to reliably detect expected fatty acids and known polyketides. In addition, up to 46 and 270 further, unknown metabolites presumably including novel polyketides were detected in the F. graminearum and T. reesei culture samples, respectively, all of which exhibited the user-predicted isotopolog patterns originating from the malonate tracer incorporation. The software can be used for every conceivable tracer approach. Furthermore, the rule sets can be easily adapted or extended if necessary. CPExtract is available free of charge for noncommercial use at https://metabolomics-ifa.boku.ac.at/CPExtract.

DOI: https://doi.org/10.1021/acs.analchem.1c04530
J Exp Bot. 2022 Feb 17. pii: erac062. [Epub ahead of print]

An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids.

Somnath Koley, Kevin L Chu, Saba S Gill, Doug K Allen.

  Assessing central carbon metabolism in plants can be challenging due to the dynamic range in pool sizes, with low levels of important phosphorylated sugars relative to more abundant sugars and organic acids. Here, we report a sensitive liquid chromatography-mass spectrometry (LC-MS) method for analyzing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The LC method was developed for enhanced selectivity of 27 central metabolites in a single run with sensitivity at femtomole levels observed for most phosphorylated sugars. The method resolved phosphorylated hexose, pentose, and triose isomers that are otherwise challenging. Compared to a standard HILIC approach, these metabolites had improved peak areas using our approach due to ion-enhancement or low ion-suppression in the biological sample matrix. The approach was applied to investigate metabolism in high lipid-producing tobacco leaves that exhibited increased levels of the acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating 13C-labeled seeds from Camelina sativa. The method provides a means to analyze intermediates more comprehensively in central metabolism of plant tissues.

Keywords:  Central metabolism; ion suppression and enhancement; isomer separation; isotopic labeling; liquid chromatography-mass spectrometry; metabolite quantification; mixed-mode column chromatography; oilseeds

DOI:  https://doi.org/10.1093/jxb/erac062
Mol Metab. 2022 Feb 09. pii: S2212-8778(22)00026-6. [Epub ahead of print] 101457

Dietary PUFA drives diverse systems-level changes in lipid metabolism.

Samuel Furse, Samuel Virtue, Stuart G Snowden, Antonio Vidal-Puig, Philip C Stevenson, Davide Chiarugi, Albert Koulman.

OBJECTIVE: Poly-unsaturated fatty acid (PUFA) supplements have been trialled as a treatment for a number of conditions, and produced a variety of results. This variety is ascribed to both the supplements, often comprising mixtures of fatty acids and to different effects in different organs. Here, we tested the hypothesis that supplementation of individual PUFAs has diverse system-level effects that are dependent on the molecular structure of the PUFA.
METHODS: We undertook a network analysis using Lipid Traffic Analysis to identify both local and systems-level changes in lipid metabolism using publicly available lipidomics data from a mouse model of supplementation with FA(20:4n-6), FA(20:5n-3) and FA(22:6n-3); arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, respectively. Lipid Traffic Analysis is a new bioinformatics tool that uses the spatial distribution of lipids to pinpoint changes or differences in control of metabolism, thereby suggesting mechanistic reasons for differences in observed lipid metabolism.
RESULTS: There was strong evidence for changes to lipid metabolism being dependent on the structure of the supplemented PUFA. Phosphatidylcholine and triglycerides showed a change in the variety more than the number of variables, whereas phosphatidylethanolamine and phosphatidylinositol showed considerable change in both which variables and the number of them, in a highly PUFA-dependent manner. There was also evidence for changes to endogenous biosynthesis of fatty acids and to both elongation and desaturation of fatty acids.
CONCLUSIONS: These results show that the full biological impact of PUFA supplementation is far wider than any single-organ effect and implies that supplementation and dosing with PUFAs requires a system-level assessment.

DOI: https://doi.org/10.1016/j.molmet.2022.101457
Int J Mol Sci. 2022 Feb 08. pii: 1919. [Epub ahead of print]23(3):

Interactions between Radiation and One-Carbon Metabolism.

Navyateja Korimerla, Daniel R Wahl.

  Metabolic reprogramming is a hallmark of cancer. Cancer cells rewire one-carbon metabolism, a central metabolic pathway, to turn nutritional inputs into essential biomolecules required for cancer cell growth and maintenance. Radiation therapy, a common cancer therapy, also interacts and alters one-carbon metabolism. This review discusses the interactions between radiation therapy, one-carbon metabolism and its component metabolic pathways.

Keywords:  cancer therapy; folate cycle; methionine cycle; one-carbon metabolism; radiation therapy

DOI:  https://doi.org/10.3390/ijms23031919
J Cheminform. 2022 Feb 16. 14(1): 6

Reproducible untargeted metabolomics workflow for exhaustive MS2 data acquisition of MS1 features.

Miao Yu, Georgia Dolios, Lauren Petrick.

  Unknown features in untargeted metabolomics and non-targeted analysis (NTA) are identified using fragment ions from MS/MS spectra to predict the structures of the unknown compounds. The precursor ion selected for fragmentation is commonly performed using data dependent acquisition (DDA) strategies or following statistical analysis using targeted MS/MS approaches. However, the selected precursor ions from DDA only cover a biased subset of the peaks or features found in full scan data. In addition, different statistical analysis can select different precursor ions for MS/MS analysis, which make the post-hoc validation of ions selected following a secondary analysis impossible for precursor ions selected by the original statistical method. Here we propose an automated, exhaustive, statistical model-free workflow: paired mass distance-dependent analysis (PMDDA), for reproducible untargeted mass spectrometry MS2 fragment ion collection of unknown compounds found in MS1 full scan. Our workflow first removes redundant peaks from MS1 data and then exports a list of precursor ions for pseudo-targeted MS/MS analysis on independent peaks. This workflow provides comprehensive coverage of MS2 collection on unknown compounds found in full scan analysis using a "one peak for one compound" workflow without a priori redundant peak information. We compared pseudo-spectra formation and the number of MS2 spectra linked to MS1 data using the PMDDA workflow to that obtained using CAMERA and RAMclustR algorithms. More annotated compounds, molecular networks, and unique MS/MS spectra were found using PMDDA compared with CAMERA and RAMClustR. In addition, PMDDA can generate a preferred ion list for iterative DDA to enhance coverage of compounds when instruments support such functions. Finally, compounds with signals in both positive and negative modes can be identified by the PMDDA workflow, to further reduce redundancies. The whole workflow is fully reproducible as a docker image xcmsrocker with both the original data and the data processing template.

Keywords:  Data analysis; High-resolution mass spectrometry; Metabolomics; Open science; Reproducible research; Workflow

DOI:  https://doi.org/10.1186/s13321-022-00586-8
Methods Mol Biol. 2022 ;2471 111-121

Differential Proteomic Analysis of Complex Mixtures by Label-Free nLC MS/MS.

Iraide Escobés, Mikel Azkargorta, Ibon Iloro, Felix Elortza.

  After over two decades of constant evolution, proteomics can be truly considered nowadays as a high-throughput technique. Latest advances performed in sample preparation, instrumentation, and data analysis tools enable proteome-wide detection and quantification of proteins in complex samples.Label-free quantification by nanoscale liquid chromatography coupled online to tandem mass spectrometry (nLC MS /MS ) is a straightforward procedure for relative protein quantification. This approach allows to get deeper insights of what molecular changes are involved in the biological system we want to study in an unbiased manner.This chapter describes methods for sample preparation prior to mass spectrometry analysis. Besides, we describe a standard acquisition method, and some common bioinformatics analyses that help extracting biologically relevant information out of the achieved data.

Keywords:  Biomarker discovery; Label-free quantification; Mass spectrometry; Precision-medicine; Proteome; Proteomics; Relative protein quantification

DOI:  https://doi.org/10.1007/978-1-0716-2193-6_5
Anal Chem. 2022 Feb 18.

Automated Annotation of Untargeted All-Ion Fragmentation LC-MS Metabolomics Data with MetaboAnnotatoR.

Gonçalo Graça, Yuheng Cai, Chung-Ho E Lau, Panagiotis A Vorkas, Matthew R Lewis, Elizabeth J Want, David Herrington, Timothy M D Ebbels.

Untargeted metabolomics and lipidomics LC-MS experiments produce complex datasets, usually containing tens of thousands of features from thousands of metabolites whose annotation requires additional MS/MS experiments and expert knowledge. All-ion fragmentation (AIF) LC-MS/MS acquisition provides fragmentation data at no additional experimental time cost. However, analysis of such datasets requires reconstruction of parent-fragment relationships and annotation of the resulting pseudo-MS/MS spectra. Here, we propose a novel approach for automated annotation of isotopologues, adducts, and in-source fragments from AIF LC-MS datasets by combining correlation-based parent-fragment linking with molecular fragment matching. Our workflow focuses on a subset of features rather than trying to annotate the full dataset, saving time and simplifying the process. We demonstrate the workflow in three human serum datasets containing 599 features manually annotated by experts. Precision and recall values of 82-92% and 82-85%, respectively, were obtained for features found in the highest-rank scores (1-5). These results equal or outperform those obtained using MS-DIAL software, the current state of the art for AIF data annotation. Further validation for other biological matrices and different instrument types showed variable precision (60-89%) and recall (10-88%) particularly for datasets dominated by nonlipid metabolites. The workflow is freely available as an open-source R package, MetaboAnnotatoR, together with the fragment libraries from Github (https://github.com/gggraca/MetaboAnnotatoR).

DOI: https://doi.org/10.1021/acs.analchem.1c03032
Bone Res. 2022 Feb 15. 10(1): 14

De novo serine synthesis regulates chondrocyte proliferation during bone development and repair.

Steve Stegen, Shauni Loopmans, Ingrid Stockmans, Karen Moermans, Peter Carmeliet, Geert Carmeliet.

The majority of the mammalian skeleton is formed through endochondral ossification starting from a cartilaginous template. Cartilage cells, or chondrocytes, survive, proliferate and synthesize extracellular matrix in an avascular environment, but the metabolic requirements for these anabolic processes are not fully understood. Here, using metabolomics analysis and genetic in vivo models, we show that maintaining intracellular serine homeostasis is essential for chondrocyte function. De novo serine synthesis through phosphoglycerate dehydrogenase (PHGDH)-mediated glucose metabolism generates nucleotides that are necessary for chondrocyte proliferation and long bone growth. On the other hand, dietary serine is less crucial during endochondral bone formation, as serine-starved chondrocytes compensate by inducing PHGDH-mediated serine synthesis. Mechanistically, this metabolic flexibility requires ATF4, a transcriptional regulator of amino acid metabolism and stress responses. We demonstrate that both serine deprivation and PHGDH inactivation enhance ATF4 signaling to stimulate de novo serine synthesis and serine uptake, respectively, and thereby prevent intracellular serine depletion and chondrocyte dysfunction. A similar metabolic adaptability between serine uptake and de novo synthesis is observed in the cartilage callus during fracture repair. Together, the results of this study reveal a critical role for PHGDH-dependent serine synthesis in maintaining intracellular serine levels under physiological and serine-limited conditions, as adequate serine levels are necessary to support chondrocyte proliferation during endochondral ossification.

DOI: https://doi.org/10.1038/s41413-021-00185-7
J Proteome Res. 2022 Feb 15.

Proteomic Analysis of Whole Blood Using Volumetric Absorptive Microsampling for Precision Medicine Biomarker Studies.

Mark P Molloy, Cameron Hill, Matthew B O'Rourke, Jason Chandra, Pascal Steffen, Matthew J McKay, Dana Pascovici, Ben R Herbert.

  Microsampling of patient blood promises several benefits over conventional phlebotomy practices to facilitate precision medicine studies. These include at-home patient blood collection, supporting telehealth monitoring, minimal postcollection processing, and compatibility with nonrefrigerated transport and storage. However, for proteomic biomarker studies, mass spectrometry of whole blood has generally been avoided in favor of using plasma or serum obtained from venepuncture. We evaluated the use of a volumetric absorptive microsampling (VAMS) device as a sample preparation matrix to enable LC-MS proteomic analyses of dried whole blood. We demonstrated the detection and robust quantitation of up to 1600 proteins from single-shot shotgun-LC-MS analysis of dried whole blood, greatly enhancing proteome depth compared with conventional single-shot LC-MS analyses of undepleted plasma. Some proteins not previously reported in blood were detected using this approach. Various washing reagents were used to demonstrate that proteins can be preferentially removed from VAMS devices prior to downstream analyses. We provide a demonstration that archival frozen blood cell pellets housed under long-term storage (exceeding 5 years) are compatible with VAMS to enable quantitation of potential biomarker proteins from biobank repositories. These demonstrations are important steps in establishing viable analysis workflows to underpin large-scale precision medicine studies. Data are available via ProteomeXchange with the identifier PXD028605.

Keywords:  blood; mass spectrometry; microsampling; precision medicine; proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.1c00971
J Clin Med. 2022 Jan 29. pii: 721. [Epub ahead of print]11(3):

Changing Metabolic Patterns along the Colorectal Adenoma-Carcinoma Sequence.

Julia Tevini, Sebastian K Eder, Ursula Huber-Schönauer, David Niederseer, Georg Strebinger, Johanna M Gostner, Elmar Aigner, Christian Datz, Thomas K Felder.

  Colorectal cancer (CRC) is a major public health burden and one of the leading causes of cancer-related deaths worldwide. Screening programs facilitate early diagnosis and can help to reduce poor outcomes. Serum metabolomics can extract vital molecular information that may increase the sensitivity and specificity of colonoscopy in combination with histopathological examination. The present study identifies serum metabolite patterns of treatment-naïve patients, diagnosed with either advanced adenoma (AA) or CRC in colonoscopy screenings, in the framework of the SAKKOPI (Salzburg Colon Cancer Prevention Initiative) program. We used a targeted flow injection analysis and liquid chromatography-tandem mass spectrometry metabolomics approach (FIA- and LC-MS/MS) to characterise the serum metabolomes of an initial screening cohort and two validation cohorts (in total 66 CRC, 76 AA and 93 controls). The lipidome was significantly perturbed, with a proportion of lipid species being downregulated in CRC patients, as compared to AA and controls. The predominant alterations observed were in the levels of lyso-lipids, glycerophosphocholines and acylcarnitines, but additionally, variations in the quantity of hydroxylated sphingolipids could be detected. Changed amino acid metabolism was restricted mainly to metabolites of the arginine/dimethylarginine/NO synthase pathway. The identified metabolic divergences observed in CRC set the foundation for mechanistic studies to characterise biochemical pathways that become deregulated during progression through the adenoma to carcinoma sequence and highlight the key importance of lipid metabolites. Biomarkers related to these pathways could improve the sensitivity and specificity of diagnosis, as well as the monitoring of therapies.

Keywords:  adenoma; colorectal cancer; lipid metabolism; metabolomics

DOI:  https://doi.org/10.3390/jcm11030721
Semin Cancer Biol. 2022 Feb 10. pii: S1044-579X(22)00031-1. [Epub ahead of print]

Metabolic modulation of immune checkpoints and novel therapeutic strategies in cancer.

Yi Wang, Yuya Wang, Yifei Ren, Qi Zhang, Ping Yi, Chunming Cheng.

  Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) or programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1)-based immune checkpoint inhibitors (ICIs) have led to significant improvements in the overall survival of patients with certain cancers and are expected to benefit patients by achieving complete, long-lasting remissions and cure. However, some patients who receive ICIs either fail treatment or eventually develop immunotherapy resistance. The existence of such patients necessitates a deeper understanding of cancer progression, specifically nutrient regulation in the tumor microenvironment (TME), which includes both metabolic cross-talk between metabolites and tumor cells, and intracellular metabolism in immune and cancer cells. Here we review the features and behaviors of the TME and discuss the recently identified major immune checkpoints. We comprehensively and systematically summarize the metabolic modulation of tumor immunity and immune checkpoints in the TME, including glycolysis, amino acid metabolism, lipid metabolism, and other metabolic pathways, and further discuss the potential metabolism-based therapeutic strategies tested in preclinical and clinical settings. These findings will help to determine the existence of a link or crosstalk between tumor metabolism and immunotherapy, which will provide an important insight into cancer treatment and cancer research.

Keywords:  Amine acid metabolism; Fatty acid synthesis; Glycolysis; Immune checkpoint; Immunotherapy; Lipid metabolism; Tumor metabolism; Tumor microenvironment

DOI:  https://doi.org/10.1016/j.semcancer.2022.02.010
Cancers (Basel). 2022 Jan 30. pii: 725. [Epub ahead of print]14(3):

The Application of Metabolomics in Recent Colorectal Cancer Studies: A State-of-the-Art Review.

Andrew Gold, Fouad Choueiry, Ning Jin, Xiaokui Mo, Jiangjiang Zhu.

  Colorectal cancer (CRC) is a highly prevalent disease with poor prognostic outcomes if not diagnosed in early stages. Current diagnosis techniques are either highly invasive or lack sufficient sensitivity. Thus, identifying diagnostic biomarkers of CRC with high sensitivity and specificity is desirable. Metabolomics represents an analytical profiling technique with great promise in identifying such biomarkers and typically represents a close tie with the phenotype of a specific disease. We thus conducted a systematic review of studies reported from January 2012 to July 2021 relating to the detection of CRC biomarkers through metabolomics to provide a collection of knowledge for future diagnostic development. We identified thirty-seven metabolomics studies characterizing CRC, many of which provided metabolites/metabolic profile-based diagnostic models with high sensitivity and specificity. These studies demonstrated that a great number of metabolites can be differentially regulated in CRC patients compared to healthy controls, adenomatous polyps, or across stages of CRC. Among these metabolite biomarkers, especially dysregulated were certain amino acids, fatty acids, and lysophosphatidylcholines. Additionally, we discussed the contribution of the gut bacterial population to pathogenesis of CRC through their modulation to fecal metabolite pools and summarized the established links in the literature between certain microbial genera and altered metabolite levels in CRC patients. Taken together, we conclude that metabolomics presents itself as a promising and effective method of CRC biomarker detection.

Keywords:  GC-MS; LC-MS; colorectal cancers; metabolite biomarker; metabolomics

DOI:  https://doi.org/10.3390/cancers14030725
EMBO Mol Med. 2022 Feb 18. e14753

Ketone body oxidation increases cardiac endothelial cell proliferation.

Eva-Maria Weis, Patrycja Puchalska, Alisa B Nelson, Jacqueline Taylor, Iris Moll, Sana S Hasan, Matthias Dewenter, Marco Hagenmüller, Thomas Fleming, Gernot Poschet, Agnes Hotz-Wagenblatt, Johannes Backs, Peter A Crawford, Andreas Fischer.

  Blood vessel formation is dependent on metabolic adaption in endothelial cells. Glucose and fatty acids are essential substrates for ATP and biomass production; however, the metabolism of other substrates remains poorly understood. Ketone bodies are important nutrients for cardiomyocytes during starvation or consumption of carbohydrate-restrictive diets. This raises the question whether cardiac endothelial cells would not only transport ketone bodies but also consume some of these to achieve their metabolic needs. Here, we report that cardiac endothelial cells are able to oxidize ketone bodies and that this enhances cell proliferation, migration, and vessel sprouting. Mechanistically, this requires succinyl-CoA:3-oxoacid-CoA transferase, a key enzyme of ketone body oxidation. Targeted metabolite profiling revealed that carbon from ketone bodies got incorporated into tricarboxylic acid cycle intermediates as well as other metabolites fueling biomass production. Elevation of ketone body levels by a high-fat, low-carbohydrate ketogenic diet transiently increased endothelial cell proliferation in mouse hearts. Notably, in a mouse model of heart hypertrophy, ketogenic diet prevented blood vessel rarefication. This suggests a potential beneficial role of dietary intervention in heart diseases.

Keywords:  angiogenesis; endothelial cell; heart; ketogenic diet; ketone bodies

DOI:  https://doi.org/10.15252/emmm.202114753
Methods Enzymol. 2022 ;pii: S0076-6879(21)00403-1. [Epub ahead of print]663 205-234

Evaluation of endogenous peptide stereochemistry using liquid chromatography-mass spectrometry-based spiking experiments.

Baba M Yussif, James W Checco.

  Functional d-amino acid-containing peptides are endogenously found throughout nature, generated through non-ribosomal peptide synthesis or through post-translational modification of ribosome-derived peptides. Despite the high functional importance of peptide stereochemistry in biomolecular recognition, identification of amino acid residue isomerization can be challenging using most common peptide characterization workflows. Here, we describe a relatively simple approach to test hypotheses regarding the stereochemistry of endogenous peptides via liquid chromatography-mass spectrometry (LC-MS) spiking experiments with synthetic isotope-labeled peptide standards. Our protocol details the synthesis of 13C-labeled synthetic peptide diastereomers via solid-phase peptide synthesis, their purification and characterization, and LC-MS experiments to evaluate putative stereochemical assignments. This approach does not require demanding purification of the endogenous peptide and is compatible with small quantities of endogenous extracts. Overall, this procedure complements current endogenous peptide characterization methods, granting the ability to relatively quickly verify the sequence and stereochemistry in endogenous peptide diastereomers directly in complex biological extracts.

Keywords:  Diastereomers; Isotope labeling; LC-MS; Peptide; Peptidomics; Solid-phase peptide synthesis; d-amino acid

DOI:  https://doi.org/10.1016/bs.mie.2021.10.009
Anal Chim Acta. 2022 Mar 01. pii: S0003-2670(21)01231-9. [Epub ahead of print]1196 339405

Untargeted detection of the carbonyl metabolome by chemical derivatization and liquid chromatography-tandem mass spectrometry in precursor ion scan mode: Elucidation of COVID-19 severity biomarkers.

Alex Gomez-Gomez, Jose Rodríguez-Morató, Noemí Haro, Judith Marín-Corral, Joan Ramon Masclans, Oscar J Pozo.

  Metabolomics (both targeted and untargeted) has become the gold standard in biomarker discovery. Whereas targeted approaches only provide information for the selected markers, thus hampering the determination of out-of-the-box markers, the common bottleneck of untargeted metabolomics is the identification of detected biomarkers. In this study, we developed a strategy based on derivatization and LC-MS/MS detection in a precursor ion scan for the untargeted determination of a specific part of the metabolome (carbonyl-containing metabolites). The usefulness of this guided metabolomics approach has been demonstrated by elucidating carbonyl-containing biomarkers of COVID-19 severity. First, the LC-MS/MS behavior of 63 model compounds after O-benzylhydroxylamine derivatization was studied. A precursor ion scan of m/z 91 was selected as a suitable approach for the untargeted detection of carbonyl-containing metabolites. The method was able to detect ≈300 potential carbonyl-containing molecules in plasma, including mono-/di-/tricarbonylic compounds with satisfactory intra-day and inter-day repeatability and RSDs commonly <15%. Additionally, the semiquantitative nature of the precursor ion scan method was confirmed by comparison with a fully validated targeted method. The application of the guided metabolomics method to COVID-19 plasma samples revealed the presence of four potential COVID-19 severity biomarkers. Based on their LC-MS/MS behavior, these biomarkers were elucidated as 2-hydroxybutyrate, 2,3-dihydroxybutyrate, 2-oxobutyrate and 2-hydroxy-3-methylbutyrate. Their structures were confirmed by comparison with reference materials. The alterations of these biomarkers with COVID-19 severity were confirmed by a target analysis of a larger set of samples. Our results confirm that guided metabolomics is an alternative approach for the untargeted detection of selected families of metabolites; this approach can accelerate their elucidation and provide new perspectives for the establishment of health/disease biomarkers.

Keywords:  Biomarker discovery; COVID-19; Carbonyl metabolome; Liquid chromatography-tandem mass spectrometry; Precursor ion scan; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.aca.2021.339405
Int J Mol Sci. 2022 Jan 30. pii: 1609. [Epub ahead of print]23(3):

Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity.

Pan Fang, Yanlong Ji, Thomas Oellerich, Henning Urlaub, Kuan-Ting Pan.

  Protein glycosylation governs key physiological and pathological processes in human cells. Aberrant glycosylation is thus closely associated with disease progression. Mass spectrometry (MS)-based glycoproteomics has emerged as an indispensable tool for investigating glycosylation changes in biological samples with high sensitivity. Following rapid improvements in methodologies for reliable intact glycopeptide identification, site-specific quantification of glycopeptide macro- and micro-heterogeneity at the proteome scale has become an urgent need for exploring glycosylation regulations. Here, we summarize recent advances in N- and O-linked glycoproteomic quantification strategies and discuss their limitations. We further describe a strategy to propagate MS data for multilayered glycopeptide quantification, enabling a more comprehensive examination of global and site-specific glycosylation changes. Altogether, we show how quantitative glycoproteomics methods explore glycosylation regulation in human diseases and promote the discovery of biomarkers and therapeutic targets.

Keywords:  glycoproteomics; label free; mass spectrometry; quantification; stable-isotope labeling

DOI:  https://doi.org/10.3390/ijms23031609
Anal Chem. 2022 Feb 14.

Equivalent Carbon Number and Interclass Retention Time Conversion Enhance Lipid Identification in Untargeted Clinical Lipidomics.

Jake B White, Paul J Trim, Thalia Salagaras, Aaron Long, Peter J Psaltis, Johan W Verjans, Marten F Snel.

Chromatography is often used as a method for reducing sample complexity prior to analysis by mass spectrometry, and the use of retention time (RT) is becoming increasingly popular to add valuable supporting information in lipid identification. The RT of lipids with the same headgroup in reversed-phase separation can be predicted using the equivalent carbon number (ECN) model. This model describes the effects of acyl chain length and degree of saturation on lipid RT. For the first time, we have found a robust correlation in the chromatographic separation of lipids with different headgroups that share the same fatty acid motive. This relationship can be exploited to perform interclass RT conversion (IC-RTC) by building a model from RT measurements from lipid standards that allows the prediction of RT of one lipid subclass based on another. Here, we utilize ECN modeling and IC-RTC to build a glycerophospholipid RT library with 517 entries based on 136 tandem mass spectrometry-characterized lipid RTs from NIST SRM-1950 plasma and lipid standards. The library was tested on a patient cohort undergoing coronary artery bypass grafting surgery (n = 37). A total of 156 unique circulating glycerophospholipids were identified, of which 52 (1 LPG, 24 PE, 5 PG, 18 PI, and 9 PS) were detected with IC-RTC, thereby demonstrating the utility of this technique for the identification of lipid species not found in commercial standards.

DOI: https://doi.org/10.1021/acs.analchem.1c03770
J Biol Chem. 2022 Feb 15. pii: S0021-9258(22)00175-2. [Epub ahead of print] 101735

Fatty acid transport protein 2 interacts with ceramide synthase 2 to promote ceramide synthesis.

Jiyoon L Kim, Beatriz Mestre, Sergey Malitsky, Maxim Itkin, Meital Kupervaser, Anthony H Futerman.

  Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl CoAs, although a number of cytosolic proteins in the pathways of acyl CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2 or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl CoA generation will impact a wide variety of down-stream lipids, not least the SLs, which utilize two acyl CoA moieties in the initial steps of their synthesis.

Keywords:  Ceramide; ceramide synthase; dihydroceramide; fatty acid transport protein; sphingolipids

DOI:  https://doi.org/10.1016/j.jbc.2022.101735
Anal Chim Acta. 2022 Mar 08. pii: S0003-2670(22)00063-0. [Epub ahead of print]1197 339492

Model of retention time and density of gradient peak capacity for improved LC-MS method optimization: Application to metabolomics.

Fabrice Gritti, Mark David, Patrick Brothy, Matthew R Lewis.

  A general and deterministic model is derived from the fundamentals of liquid chromatography to calculate retention time, peak width, peak capacity, and density of peak capacity in gradient liquid chromatography. The calculation of these chromatographic properties accounts for 1) the presence of initial (separation of the earliest eluters) and final (column wash) isocratic steps before and after the linear gradient, respectively, 2) the pre- (flow through needle and preheater tubes) and post-column (outlet and emitter tubes before MS detection) dispersion, 3) the compression of the chromatographic band, and 4) the retention of the organic modifier onto the RPLC column. The multiple and variable method parameters may include the column dimensions, particle size, flow rate, temperature, initial and final isocratic hold times, gradient time, gradient steepness, column conditioning/sample load time, and the pre- and post-column tube dimensions. The model enables the users to perform robust multi-dimensional optimization of UHPLC-MS methods and offers the possibility to predict the expected MS feature density for increased method performance. Method optimization can be further improved by matching the observed MS feature density (number of metabolites detected as function of time) to the predicted density of peak capacity. It is directly applied to the optimization of high-throughput RPLC separation methods specifically designed for large-scale urinary metabolic phenotyping.

Keywords:  Density of peak capacity; LC-MS method optimization; Non-targeted metabolomics; Retention time and peak width prediction; Sample feature density; Urine metabolites

DOI:  https://doi.org/10.1016/j.aca.2022.339492
J Chromatogr A. 2022 Feb 02. pii: S0021-9673(22)00068-1. [Epub ahead of print]1667 462870

Accurate determination of amino acids by quadruple isotope dilution-reverse phase liquid Chromatography-Tandem mass spectrometry after derivatization with 2-Naphthoyl chloride.

Elif Öztürk Er, Sezin Erarpat, Süleyman Bodur, Ömer Tahir Günkara, Belma Özbek, Sezgin Bakırdere.

  The determination of amino acids in biological samples is central to the diagnosis of inherited metabolic disorders and also gives significant information about the metabolisms in the cells and living body. The development of analytical method for reliable quantification of amino acids in biological samples is still challenging because of the polar nature of amino acids and complex nature of biological samples causing a high degree of interferences during analysis. In the present study, a pre-column derivatization method using 2-naphtoyl chloride combined with liquid chromatography-tandem mass spectrometry method was developed for the determination of 17 amino acids in human serum and urine matrices. Low detection limits were obtained in the range of 0.015 - 0.266 µmol kg-1 and acceptable recovery results were obtained in human serum and urine samples. Isotopically labelled (15N labelled) amino acids were spiked to standards and samples before derivatization to compensate for the analytical errors in the whole procedure. The combination of quadrupole isotope dilution strategy with the derivatization based reversed phase chromatography allowed to improve method accuracy and precision.

Keywords:  Amino acids; Derivatization; Human serum; Human urine; Isotope dilution

DOI:  https://doi.org/10.1016/j.chroma.2022.462870