bims-mascan 2024-12-15 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2024–12–15
23 papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

Reproducible protein quantitation of 270 human proteins at increased depth using nanoparticle-based fractionation and multiple reaction monitoring mass spectrometry with stable isotope-labelled internal standards.
Matrix effect evaluation using multi-component post-column infusion in untargeted hydrophilic interaction liquid chromatography-mass spectrometry plasma metabolomics.
A new understanding of tissue biology from MS-based proteomics at single-cell resolution.
Tools and techniques for quantitative glycoproteomic analysis.
Deeper insights into the stability of oxylipins in human plasma across multiple freeze-thaw cycles and storage conditions.
Crosstalk between metabolic and epigenetic modifications during cell carcinogenesis.
Redesigning error control in cross-linking mass spectrometry enables more robust and sensitive protein-protein interaction studies.
Targeting pivotal amino acids metabolism for treatment of leukemia.
Chiral supercritical fluid chromatography of monoacylglycerol and diacylglycerol enantiomers in biological samples: Adjusting selectivity via column coupling.
Intact Mass Proteomics Using a Proteoform Atlas.
Metabolic reprogramming, sensing, and cancer therapy.
Reciprocal links between methionine metabolism, DNA repair and therapy resistance in glioblastoma.
Addressing Sample Mix-Ups: Tools and Approaches for Large-Scale Multi-Omics Studies.
Fast and reliable method for analysis of derivatized plasma amino acids by liquid chromatography-single quadrupole-mass spectrometry.
Bioinert UHPLC system improves sensitivity and peak shapes for ionic metabolites.
TBK1 Reprograms Metabolism in Breast Cancer: An Integrated Omics Approach.
Applications of Mass Spectrometry Proteomic Methods to Immunoglobulins in the Clinical Laboratory.
Integration of lipidomics with targeted, single cell, and spatial transcriptomics defines an unresolved pro-inflammatory state in colon cancer.
Development of a Multiplexed LC-MS/MS Assay for the Quantitation of Podocyte Injury Biomarkers Nephrin, Podocalyxin, and Podocin in Human Urine.
MeTEor: an R Shiny app for exploring longitudinal metabolomics data.
Continuous collective analysis of chemical reactions.
Carnitine palmitoyltransferase 1 facilitates fatty acid oxidation in a non-cell-autonomous manner.
Enhanced fatty acid oxidation in osteoprogenitor cells provides protection from high-fat diet induced bone dysfunction.

Analyst. 2024 Dec 13.

Reproducible protein quantitation of 270 human proteins at increased depth using nanoparticle-based fractionation and multiple reaction monitoring mass spectrometry with stable isotope-labelled internal standards.

Claudia Gaither, Robert Popp, Aaron S Gajadhar, Christoph H Borchers.

Here we show that when using a mix of 274 light synthetic peptide standards (NAT) as surrogates for 270 human plasma proteins, as well as stable isotope-labelled standards (SIS) as normalizers (both from MRM Proteomics Inc.) for targeted quantitative analysis by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS), the Seer Proteograph™ platform allowed for the enrichment and absolute quantitation of up to an additional 62 targets (median) compared to two standard proteomic workflows without enrichment, representing an increase of 44%. The nanoparticle-based fractionation workflow resulted in improved reproducibility compared to a traditional proteomic workflow with no fractionation (median 8.3% vs. 13.1% CV). As expected, the protein concentrations in nanoparticle coronas were higher and had more compressed dynamic range in comparison to the concentrations determined either by a 3-hour Trypsin/LysC or overnight tryptic digestion methods. As the nanoparticle-based fractionation technology gains popularity, additional steps such as establishing technique-specific protein reference ranges across plasma samples and comparisons to well-established protein quantitation methods like enzyme-linked immunosorbent assay (ELISA) and LC/MRM-MS may be explored to enable absolute quantification of plasma proteins based on nanoparticle-based fractionation data.

DOI: https://doi.org/10.1039/d4an00967c
J Chromatogr A. 2024 Dec 03. pii: S0021-9673(24)00953-1. [Epub ahead of print]1740 465580

Matrix effect evaluation using multi-component post-column infusion in untargeted hydrophilic interaction liquid chromatography-mass spectrometry plasma metabolomics.

Mengle Zhu, Lieke Lamont, Pascal Maas, Amy C Harms, Marian Beekman, P Eline Slagboom, Anne-Charlotte Dubbelman, Thomas Hankemeier.

  Metabolomics based on hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (MS) is a powerful tool for polar metabolite identification and quantification to further contribute to biomarker discovery and disease mechanism elucidation. However, matrix effect (ME), which may lead to altered ionization efficiency due to co-eluting compounds, is a significant challenge during biological analysis. Therefore, ME evaluation plays a crucial role during method development. Two approaches to evaluate ME are using stable isotope labelled-internal standards (SIL-IS) and post-column infusion (PCI) of standards. In this study, we developed an untargeted HILIC-MS method by applying four PCI standards for ME evaluation. We found PCI is a compelling approach for ME assessment compared to SIL-IS method due to its advantage in untargeted analysis. Through the ME evaluation and chromatographic performance comparison of 18 SIL standards across three columns and three different mobile phase pH conditions, our findings revealed that the BEH-Z-HILIC column operated at pH 4 with 10 mM ammonium formate exhibited minimal ME and superior performance. The method showed exceptional linearity (R² > 0.98), reliable repeatability (RSD < 15 %), good inter-day precision (RSD < 30 %), and acceptable recovery (>75 %) for all SIL standards. Absolute matrix effect (AME) and relative matrix effect (RME) assessment in three plasma donors revealed a high consistency between PCI and SIL-IS approaches. Finally, this method coupled with the PCI approach was applied to 40 plasma samples. Fifty endogenous compounds were detected and their AME and RME were evaluated. Results showed that many compounds experienced severe ion suppression, though their ME variation between 40 samples is low. In conclusion, PCI method is a robust alternative for monitoring ME and evaluating ME of endogenous compounds during untargeted method optimization and biological analysis.

Keywords:  HILIC; Matrix effect; Plasma; Post-column infusion; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.chroma.2024.465580
Nat Methods. 2024 Dec;21(12): 2220-2222

A new understanding of tissue biology from MS-based proteomics at single-cell resolution.

Thierry M Nordmann, Andreas Mund, Matthias Mann.

DOI: https://doi.org/10.1038/s41592-024-02541-x
Biochem Soc Trans. 2024 Dec 10. pii: BST20240257. [Epub ahead of print]

Tools and techniques for quantitative glycoproteomic analysis.

Siyuan Kong, Wei Zhang, Weiqian Cao.

  Recent advances in mass spectrometry (MS)-based methods have significantly expanded the capabilities for quantitative glycoproteomics, enabling highly sensitive and accurate quantitation of glycosylation at intact glycopeptide level. These developments have provided valuable insights into the roles of glycoproteins in various biological processes and diseases. In this short review, we summarize pertinent studies on quantitative techniques and tools for site-specific glycoproteomic analysis published over the past decade. We also highlight state-of-the-art MS-based software that facilitate multi-dimension quantification of the glycoproteome, targeted quantification of specific glycopeptides, and the analysis of glycopeptide isomers. Additionally, we discuss the potential applications of these technologies in clinical biomarker discovery and the functional characterization of glycoproteins in health and disease. The review concludes with a discussion of current challenges and future perspectives in the field, emphasizing the need for more precise, high-throughput and efficient methods to further advance quantitative glycoproteomics and its applications.

Keywords:  intact glycopeptide; mass spectrometry; quantitative glycoproteomics; quantitative tools

DOI:  https://doi.org/10.1042/BST20240257
J Pharm Biomed Anal. 2024 Nov 29. pii: S0731-7085(24)00629-0. [Epub ahead of print]255 116587

Deeper insights into the stability of oxylipins in human plasma across multiple freeze-thaw cycles and storage conditions.

Maria Moran-Garrido, Sandra M Camunas-Alberca, Jorge Sáiz, Ana Gradillas, Ameer Y Taha, Coral Barbas.

  Oxylipins are signaling lipids derived from the oxidation of polyunsaturated fatty acids (PUFAs). In lipidomic studies, human plasma may be subjected to various storage conditions and freeze-thaw cycles, which may impact the analysis of these compounds. In this study, we used liquid chromatography coupled with mass spectrometry (LC-MS) to examine the influence of up to five freeze-thaw cycles (FTCs) on free and total (mostly esterified) oxylipins in human plasma and the influence of temperature and storage duration (4 °C for up to 120 h and -20 °C and -80 °C for 1-98 days) in the presence or absence of butylated hydroxytoluene (BHT) on extracted oxylipins stored in LC-MS amber vials. In fresh plasma subjected to several FTCs, approximately 48 % of the detected free oxylipins were significantly altered by the third cycle, with increases in cytochrome P450 (CYP450) and lipoxygenase (LOX)-derived compounds and reductions in trihydroxylated oxylipins. In contrast, multiple FTCs did not significantly alter esterified oxylipins. At 4 °C, the extracted oxylipins did not change significantly for up to 120 h (5 days). Oxylipin levels remained stable for 98 days at -80 °C but decreased by 98 days at -20 °C. The antioxidant activity of butylated hydroxytoluene (BHT) did not influence oxylipin stability at 4 °C for 120 h or at -80 °C for 98 days, but it reduced oxylipin degradation at -20 °C at 98 days. Conversely, prostaglandin F2α (PGF2α) exhibited substantial increases at -20 °C and -80 °C, independent of BHT. This study demonstrates that (i) unlike free oxylipins, the esterified oxylipin pool remains stable following repeated FTCs, (ii) extracted oxylipins are stable at 4 °C for up to 120 h and at -80 °C for up to 98 days, but not at -20 °C for 98 days, and (iii) BHT may minimize oxylipin degradation of sample extracts stored at -20 °C. This study provides a framework for measuring oxylipins under various freeze-thaw and storage conditions.

Keywords:  Esterified oxylipins; Free oxylipins; Freeze-thaw; Human plasma; LC-MS/MS; Redox Lipidomics; Stability

DOI:  https://doi.org/10.1016/j.jpba.2024.116587
iScience. 2024 Dec 20. 27(12): 111359

Crosstalk between metabolic and epigenetic modifications during cell carcinogenesis.

Yue Gao, Siyu Zhang, Xianhong Zhang, Yitian Du, Ting Ni, Shuailin Hao.

  Genetic mutations arising from various internal and external factors drive cells to become cancerous. Cancerous cells undergo numerous changes, including metabolic reprogramming and epigenetic modifications, to support their abnormal proliferation. This metabolic reprogramming leads to the altered expression of many metabolic enzymes and the accumulation of metabolites. Recent studies have shown that these enzymes and metabolites can serve as substrates or cofactors for chromatin-modifying enzymes, thereby participating in epigenetic modifications and promoting carcinogenesis. Additionally, epigenetic modifications play a role in the metabolic reprogramming and immune evasion of cancer cells, influencing cancer progression. This review focuses on the origins of cancer, particularly the metabolic reprogramming of cancer cells and changes in epigenetic modifications. We discuss how metabolites in cancer cells contribute to epigenetic remodeling, including lactylation, acetylation, succinylation, and crotonylation. Finally, we review the impact of epigenetic modifications on tumor immunity and the latest advancements in cancer therapies targeting these modifications.

Keywords:  Epigenetics; Molecular genetics

DOI:  https://doi.org/10.1016/j.isci.2024.111359
Mol Syst Biol. 2024 Dec 09.

Redesigning error control in cross-linking mass spectrometry enables more robust and sensitive protein-protein interaction studies.

Boris Bogdanow, Max Ruwolt, Julia Ruta, Lars Mühlberg, Cong Wang, Wen-Feng Zeng, Arne Elofsson, Fan Liu.

  Cross-linking mass spectrometry (XL-MS) allows characterizing protein-protein interactions (PPIs) in native biological systems by capturing cross-links between different proteins (inter-links). However, inter-link identification remains challenging, requiring dedicated data filtering schemes and thorough error control. Here, we benchmark existing data filtering schemes combined with error rate estimation strategies utilizing concatenated target-decoy protein sequence databases. These workflows show shortcomings either in sensitivity (many false negatives) or specificity (many false positives). To ameliorate the limited sensitivity without compromising specificity, we develop an alternative target-decoy search strategy using fused target-decoy databases. Furthermore, we devise a different data filtering scheme that takes the inter-link context of the XL-MS dataset into account. Combining both approaches maintains low error rates and minimizes false negatives, as we show by mathematical simulations, analysis of experimental ground-truth data, and application to various biological datasets. In human cells, inter-link identifications increase by 75% and we confirm their structural accuracy through proteome-wide comparisons to AlphaFold2-derived models. Taken together, target-decoy fusion and context-sensitive data filtering deepen and fine-tune XL-MS-based interactomics.

Keywords:  Cross-linking Mass Spectrometry; Error Control; False-Discovery Rate; Proteomics; Structure Modeling

DOI:  https://doi.org/10.1038/s44320-024-00079-w
Heliyon. 2024 Dec 15. 10(23): e40492

Targeting pivotal amino acids metabolism for treatment of leukemia.

Jiankun Hong, Wuling Liu, Xiao Xiao, Babu Gajendran, Yaacov Ben-David.

  Metabolic reprogramming is a crucial characteristic of cancer, allowing cancer cells to acquire metabolic properties that support their survival, immune evasion, and uncontrolled proliferation. Consequently, targeting cancer metabolism has become an essential therapeutic strategy. Abnormal amino acid metabolism is not only a key aspect of metabolic reprogramming but also plays a significant role in chemotherapy resistance and immune evasion, particularly in leukemia. Changes in amino acid metabolism in tumor cells are typically driven by a combination of signaling pathways and transcription factors. Current approaches to targeting amino acid metabolism in leukemia include inhibiting amino acid transporters, blocking amino acid biosynthesis, and depleting specific amino acids to induce apoptosis in leukemic cells. Different types of leukemic cells rely on the exogenous supply of specific amino acids, such as asparagine, glutamine, arginine, and tryptophan. Therefore, disrupting the supply of these amino acids may represent a vulnerability in leukemia. This review focuses on the pivotal role of amino acids in leukemia metabolism, their impact on leukemic stem cells, and their therapeutic potential.

Keywords:  Amino-acid metabolism; Amino-acid transporters; Chemo-resistance; Immune invasion; Leukemia; Leukemic stem cells; Metabolic reprogramming; Signaling pathways

DOI:  https://doi.org/10.1016/j.heliyon.2024.e40492
J Chromatogr A. 2024 Dec 09. pii: S0021-9673(24)00963-4. [Epub ahead of print]1740 465591

Chiral supercritical fluid chromatography of monoacylglycerol and diacylglycerol enantiomers in biological samples: Adjusting selectivity via column coupling.

Oleksandr Kozlov, Nela Štěrbová, Miroslav Lísa.

  The distinction of lipid isomers is gaining more attention in lipidomics due to their different biochemical properties in the organism. Herein, we aimed to develop a method for the analysis of monoacylglycerol (MG) and diacylglycerol (DG) enantiomers in biological samples using chiral supercritical fluid chromatography and mass spectrometry (SFC-MS). Amylose-based chiral columns showed a certain degree of separation of MG and DG isomers, but low selectivity for the acylglycerol classes in total lipid extracts, which could not be improved by modifier composition or other chromatographic conditions. The coelution of MG and DG enantiomers with highly concentrated triacylglycerols (TGs) negatively affected their MS determination based on the peak area ratio, therefore the interclass selectivity of chiral SFC was adjusted by coupling with an achiral column. The connection of the amylose tris-(3,5-dimethylphenylcarbamate) chiral column with octadecyl achiral column using a methanol as a modifier provided an excellent interclass separation of acylglycerols with the resolution of 5.53 and 15.17 for oleic acid-based MG/DG and DG/TG classes, respectively. The developed method enabled the determination of MG and DG enantiomers in complex total lipid extracts of biological samples in a 15 min gradient without time-consuming sample prefractionation. Chiral SFC-MS analysis of egg yolk, human plasma, and porcine brain samples showed different ratios of enantiomers, suggesting their unique roles within each sample type.

Keywords:  Chiral separation; Column coupling; Diacylglycerol; Monoacylglycerol; Selectivity; Supercritical fluid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2024.465591
J Proteome Res. 2024 Dec 11.

Intact Mass Proteomics Using a Proteoform Atlas.

John G Pavek, Isabella T Whitworth, Lisa Nakayama, Mark Scalf, Brian L Frey, Lloyd M Smith.

  Top-down proteomics, the characterization of intact proteoforms by tandem mass spectrometry, is the principal method for proteoform characterization in complex samples. Top-down proteomics relies on precursor isolation and subsequent gas-phase fragmentation to make proteoform identifications. While this strategy can produce highly detailed molecular information, the reliance on time-intensive tandem MS limits the speed with which proteoforms can be identified. We suggest that once proteoforms have been identified by top-down analysis in a system of interest, and archived in a system-specific Proteoform Atlas, subsequent analyses in that system can utilize the Atlas information to enable simpler and faster MS1-only identifications. We explore this idea here, using the E. coli ribosome as a model system of limited complexity. We used deep top-down analysis to construct an E. coli ribosomal Proteoform Atlas containing 2099 proteoforms from 52 of the 54 proteins that make up the E. coli ribosome. We show that using the Atlas enables confident MS1-only identifications of E. coli ribosomal proteoforms from E. coli that were perturbed by exposure to cold. Furthermore, this Atlas strategy identifies proteoforms up to 77% more rapidly compared to top-down identifications that require acquisition of both MS1 and MS2 spectra.

Keywords:  MS1; atlas; efficiency; intact-mass; proteoform; proteoforms; top-down

DOI:  https://doi.org/10.1021/acs.jproteome.4c00838
Cell Rep. 2024 Dec 12. pii: S2211-1247(24)01415-3. [Epub ahead of print]43(12): 115064

Metabolic reprogramming, sensing, and cancer therapy.

Youxiang Mao, Ziyan Xia, Wenjun Xia, Peng Jiang.

  The metabolic reprogramming of tumor cells is a crucial strategy for their survival and proliferation, involving tissue- and condition-dependent remodeling of certain metabolic pathways. While it has become increasingly clear that tumor cells integrate extracellular and intracellular signals to adapt and proliferate, nutrient and metabolite sensing also exert direct or indirect influences, although the underlying mechanisms remain incompletely understood. Furthermore, metabolic changes not only support the rapid growth and dissemination of tumor cells but also promote immune evasion by metabolically "educating" immune cells in the tumor microenvironment (TME). Recent studies have highlighted the profound impact of metabolic reprogramming on the TME and the potential of targeting metabolic pathways as a therapeutic strategy, with several enzyme inhibitors showing promising results in clinical trials. Thus, understanding how tumor cells alter their metabolic pathways and metabolically remodel the TME to support their survival and proliferation may offer new strategies for metabolic therapy and immunotherapy.

Keywords:  CP: Metabolism; immunometabolism; metabolic reprogramming; metabolite sensing; tumor metabolism; tumor therapy

DOI:  https://doi.org/10.1016/j.celrep.2024.115064
bioRxiv. 2024 Nov 21. pii: 2024.11.20.624542. [Epub ahead of print]

Reciprocal links between methionine metabolism, DNA repair and therapy resistance in glioblastoma.

Navyateja Korimerla, Baharan Meghdadi, Isra Haq, Kari Wilder-Romans, Jie Xu, Nicole Becker, Ziqing Zhu, Peter Kalev, Nathan Qi, Charles Evans, Maureen Kachman, Zitong Zhao, Angelica Lin, Andrew J Scott, Alexandra O'Brien, Ayesha Kothari, Peter Sajjakulnukit, Li Zhang, Sravya Palavalasa, Erik R Peterson, Marc L Hyer, Katya Marjon, Taryn Sleger, Meredith A Morgan, Costas A Lyssiotis, Everett M Stone, Sean P Ferris, Theodore S Lawrence, Deepak Nagrath, Weihua Zhou, Daniel R Wahl.

Glioblastoma (GBM) is uniformly lethal due to profound treatment resistance. Altered cellular metabolism is a key mediator of GBM treatment resistance. Uptake of the essential sulfur-containing amino acid methionine is drastically elevated in GBMs compared to normal cells, however, it is not known how this methionine is utilized or whether it relates to GBM treatment resistance. Here, we find that radiation acutely increases the levels of methionine-related metabolites in a variety of treatment-resistant GBM models. Stable isotope tracing studies further revealed that radiation acutely activates methionine to S-adenosyl methionine (SAM) conversion through an active signaling event mediated by the kinases of the DNA damage response. In vivo tumor SAM synthesis increases after radiation, while normal brain SAM production remains unchanged, indicating a tumor- specific metabolic alteration to radiation. Pharmacological and dietary strategies to block methionine to SAM conversion slowed DNA damage response and increased cell death following radiation in vitro. Mechanistically, these effects are due to depletion of DNA repair proteins and are reversed by SAM supplementation. These effects are selective to GBMs lacking the methionine salvage enzyme methylthioadenosine phosphorylase. Pharmacological inhibition of SAM synthesis hindered tumor growth in flank and orthotopic in vivo GBM models when combined with radiation. By contrast, methionine depletion does not reduce tumor SAM levels and fails to radiosensitize intracranial models, indicating depleting SAM, as opposed to simply lowering methionine, is critical for hindering tumor growth in intracranial models of GBM. These results highlight a new signaling link between DNA damage and SAM synthesis and define the metabolic fates of methionine in GBM in vivo . Inhibiting radiation-induced SAM synthesis slows DNA repair and augments radiation efficacy in GBM. Using MAT2A inhibitors to deplete SAM may selectively overcome treatment resistance in GBMs with defective methionine salvage while sparing normal brain.

DOI: https://doi.org/10.1101/2024.11.20.624542
Proteomics. 2024 Dec 10. e202400271

Addressing Sample Mix-Ups: Tools and Approaches for Large-Scale Multi-Omics Studies.

Yingxue Fu, Zuo-Fei Yuan, Long Wu, Junmin Peng, Xusheng Wang, Anthony A High.

  Advances in high-throughput omics technologies have enabled system-wide characterization of biological samples across multiple molecular levels, such as the genome, transcriptome, and proteome. However, as sample sizes rapidly increase in large-scale multi-omics studies, sample mix-ups have become a prevalent issue, compromising data integrity and leading to erroneous conclusions. The interconnected nature of multi-omics data presents an opportunity to identify and correct these errors. This review examines the potential sources of sample mix-ups and evaluates the methodologies and tools developed for detecting and correcting these errors, with an emphasis on approaches applicable to proteomics data. We categorize existing tools into three main groups: expression/protein quantitative trait loci-based, genotype concordance-based, and gene/protein expression correlation-based approaches. Notably, only a handful of tools currently utilize the proteogenomics approach for correcting sample mix-ups at the proteomics level. Integrating the strengths of current tools across diverse data types could enable the development of more versatile and comprehensive solutions. In conclusion, verifying sample identity is a critical first step to reduce bias and increase precision in subsequent analyses for large-scale multi-omics studies. By leveraging these tools for identifying and correcting sample mix-ups, researchers can significantly improve the reliability and reproducibility of biomedical research.

Keywords:  bioinformatics; multi‐omics; proteogenomics; proteomics; sample mix‐ups

DOI:  https://doi.org/10.1002/pmic.202400271
JDS Commun. 2024 Nov;5(6): 745-750

Fast and reliable method for analysis of derivatized plasma amino acids by liquid chromatography-single quadrupole-mass spectrometry.

August Hoppmann, Sebastian I Arriola Apelo.

The pool of free, genetically encoded AA in plasma plays an essential role not only as substrate for every protein synthesized in the body but also as signaling molecules that regulate a wide range of physiological processes. Here we present a method for the analysis of 19 of the 20 encoded AA (except Cys) in dairy cow plasma. Isolated plasma or standards for the 19 AA were gravimetrically mixed with an internal standard mix consisting of 13C isotopes of each AA. Plasma proteins were precipitated on acetonitrile and supernatants transferred to glass vials. For precolumn derivatization, plasma supernatants were buffered with sodium borate (pH 9.5-10), and AA were derivatized with 9-fluorenylmethoxycarbonyl (Fmoc) chloride. Analytes were isolated by solid-phase extraction using a strong-anionic ion exchange column and dry eluates were reconstituted in mobile phase consisting of 70% water solution of ammonium formate and 30% acetonitrile. Amino acid derivatives were separated by reverse-phase liquid chromatography over 17.5 min with a C18 column in which acetonitrile increased to 80% over the first 11 min of the method, before returning to initial levels. Electrospray ionization on negative mode was used for most AA, except Arg and Pro, for which positive mode yielded superior results. Single or double (only Lys) derivatives were measured by single quadrupole-mass spectrometry. We hypothesized that precolumn Fmoc derivatization would yield optimal resolution for quantitative analysis of the 19 targeted AA and their respective 13C internal standards, with limits of quantitation beyond physiological ranges. All 19 AA were detected with minimal background noise. An 11-point standard curve was developed for each AA. Limits of quantitation were beyond concentrations observed in plasma samples of lactating dairy cows, except for Gly where upper curve points had to be removed to maintain linearity, limiting quantitation to the upper range of physiological concentration. After removing the 4 highest concentrations from the Gly standard curve, coefficients of determination were greater than 0.999 for all of the AA. Recovery of spiked AA from plasma samples ranged from 89.9% for Phe to 100.3% for Trp. Instrument repeatability averaged 0.91 and ranged from 0.33 for Val to 2.29 for Arg. Meanwhile, sample preparation method repeatability averaged 2.02 and ranged from 1.14 for Tyr to 3.34 for Arg. Although robust methods have been developed, they depend on either availability of sophisticated instruments, mostly limited to core facilities (i.e., tandem MS methods), long and expensive chromatography without specific internal standards for each AA (i.e., HPLC-ultraviolet and HPLC-fluorescence detector), or unstable derivatization (GC-MS). Here we describe a method with high throughput, stable derivatization, high precision and recovery, and potentially more affordable than most existing methods. This method could help dairy nutritionists to consider plasma AA information for diet formulation strategies, potentially reducing feeding costs and N emissions.

DOI: https://doi.org/10.3168/jdsc.2024-0546
J Chromatogr A. 2024 Dec 06. pii: S0021-9673(24)00960-9. [Epub ahead of print]1740 465588

Bioinert UHPLC system improves sensitivity and peak shapes for ionic metabolites.

Ondřej Peterka, Alena Langová, Robert Jirásko, Michal Holčapek.

  The analysis of ionic compounds by liquid chromatography is challenging due to the interaction of analytes with the metal surface of the instrument and the column, leading to poor peak shape and decreased sensitivity. The use of bioinert materials in the chromatographic system minimizes these unrequired interactions. In this work, the ultrahigh-performance liquid chromatography (UHPLC) with bioinert components was connected to a high-resolution mass spectrometer to develop a method for untargeted metabolomic analysis. 81 standards of metabolites were used for the development and optimization of the method. In comparison to the conventional chromatographic system, the application of bioinert technology resulted in significantly improved peak shapes and increased sensitivity, especially for metabolites containing phosphate groups. The calibration curves were constructed for the evaluation of the method performance, showing a wide dynamic range, low limit of detection, and linear regression coefficients higher than 0.99 for all standards. The optimized method was applied to the analysis of NIST SRM 1950 human plasma, which allowed the detection of 156 metabolites and polar lipids based on the combination of mass accuracy in the full-scan mass spectra in both polarity modes, characteristic fragment ions in MS/MS, and logical chromatographic behavior leading to the high confidence level of annotation/identification. We have demonstrated an improvement in the peak shapes and sensitivity of ionic metabolites using bioinert technology, which indicates the potential for the analysis of other ionic compounds, e.g., molecules containing phosphate groups.

Keywords:  Bioinert system; Human plasma; Mass spectrometry; Metabolomics; Ultrahigh-performance liquid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2024.465588
J Proteome Res. 2024 Dec 13.

TBK1 Reprograms Metabolism in Breast Cancer: An Integrated Omics Approach.

Meenu Maan, Neha Jaiswal, Min Liu, Harold I Saavedra, Srikumar P Chellappan, Mainak Dutta.

  Metabolic rewiring is required for cancer cells to survive in harsh microenvironments and is considered to be a hallmark of cancer. Specific metabolic adaptations are required for a tumor to become invasive and metastatic. Cell division and metabolism are inherently interconnected, and several cell cycle modulators directly regulate metabolism. Here, we report that TBK1, which is a noncanonical IKK kinase with known roles in cell cycle regulation and TLR signaling, affects cellular metabolism in cancer cells. While TBK1 is reported to be overexpressed in several cancers and its enhanced protein level correlates with poor prognosis, the underlying molecular mechanism involved in the tumor-promoting role of TBK1 is not fully understood. In this study, we show a novel role of TBK1 in regulating cancer cell metabolism using combined metabolomics, transcriptomics, and pharmacological approaches. We find that TBK1 mediates the regulation of nucleotide and energy metabolism through aldo-keto reductase B10 (AKRB10) and thymidine phosphorylase (TYMP) genes, suggesting that this TBK1-mediated metabolic rewiring contributes to its oncogenic function. In addition, we find that TBK1 inhibitors can act synergistically with AKRB10 and TYMP inhibitors to reduce cell viability. These findings raise the possibility that combining these inhibitors might be beneficial in combating cancers that show elevated levels of TBK1.

Keywords:  AKR1B10; TBK1; TYMP; cancer metabolism; drug synergy; metabolomics; omics

DOI:  https://doi.org/10.1021/acs.jproteome.4c00530
Clin Chem. 2024 Dec 02. 70(12): 1422-1435

Applications of Mass Spectrometry Proteomic Methods to Immunoglobulins in the Clinical Laboratory.

David L Murray, Maria A V Willrich.

BACKGROUND: Immunoglobulin (Ig) measurements in the clinical laboratory have been traditionally performed by nephelometry, turbidimetry, electrophoresis, and ELISA assays. Mass spectrometry (MS) measurements have the potential to provide deeper insights on the nature of these markers.
CONTENT: Different approaches-top-down, middle-down, or bottom-up-have been described for measuring specific Igs for endogenous monoclonal immunoglobulins (M-proteins) and exogenous therapeutic monoclonal antibody therapies (t-mAbs). Challenges arise in distinguishing the Ig of interest from the polyclonal Ig background. MS is emerging as a practical method to provide quantitative analysis and information about structural and clonal features that are not easily determined by current clinical laboratory methods. This review discusses clinically implemented examples, including isotyping and quantification of M-proteins and quantitation of t-mAbs within the polyclonal Ig background, as examples of how MS can enhance our detection and characterization of Igs.
SUMMARY: This review of current clinically available MS proteomic tests for Igs highlights both analytical and nonanalytical challenges for implementation. Given the new insight into Igs from these methods, it is hoped that vendors, laboratorians, healthcare providers, and payment systems can work to overcome these challenges and advance the care of patients.

DOI: https://doi.org/10.1093/clinchem/hvae179
Gut. 2024 Dec 10. pii: gutjnl-2024-332535. [Epub ahead of print]

Integration of lipidomics with targeted, single cell, and spatial transcriptomics defines an unresolved pro-inflammatory state in colon cancer.

Ramani Soundararajan, Michelle M Maurin, Jetsen Rodriguez-Silva, Gunjan Upadhyay, Ashley J Alden, Siddabasave Gowda B Gowda, Michael J Schell, Mingli Yang, Noah Jhad Levine, Divyavani Gowda, Punith M Sundaraswamy, Shu-Ping Hui, Lance Pflieger, Heiman Wang, Jorge Marcet, Carolina Martinez, Robert David Bennett, Allen Chudzinski, Andreas Karachristos, Timothy M Nywening, Paul M Cavallaro, Matthew Linley Anderson, Robert J Coffey, Michael V Nebozhyn, Andrey Loboda, Domenico Coppola, Warren Jackson Pledger, Ganesh Halade, Timothy J Yeatman.

   BACKGROUND: Over a century ago, Virchow proposed that cancer represents a chronically inflamed, poorly healing wound. Normal wound healing is represented by a transitory phase of inflammation, followed by a pro-resolution phase, with prostaglandin (PGE2/PGD2)-induced 'lipid class switching' producing inflammation-quenching lipoxins (LXA4, LXB4).
OBJECTIVE: We explored if lipid dysregulation in colorectal cancers (CRCs) is driven by a failure to resolve inflammation.
DESIGN: We performed liquid chromatography and tandem mass spectrometry (LC-MS/MS) untargeted analysis of 40 human CRC and normal paired samples and targeted, quantitative analysis of 81 human CRC and normal paired samples. We integrated analysis of lipidomics, quantitative reverse transcription-PCR, large scale gene expression, and spatial transcriptomics with public scRNASEQ data to characterize pattern, expression and cellular localisation of genes that produce and modify lipid mediators.
RESULTS: Targeted, quantitative LC-MS/MS demonstrated a marked imbalance of pro-inflammatory mediators, with a dearth of resolving lipid mediators. In tumours, we observed prominent over-expression of arachidonic acid derivatives, the genes encoding their synthetic enzymes and receptors, but poor expression of genes producing pro-resolving synthetic enzymes and resultant lipoxins (LXA4, LXB4) and associated receptors. These results indicate that CRC is the product of defective lipid class switching likely related to inadequate or ineffective levels of PGE2/PGD2.
CONCLUSION: We show that the lipidomic profile of CRC tumours exhibits a distinct pro-inflammatory bias with a deficiency of endogenous resolving mediators secondary to defective lipid class switching. These observations pave the way for 'resolution medicine', a novel therapeutic approach for inducing or providing resolvins to mitigate the chronic inflammation driving cancer growth and progression.

Keywords:  COLORECTAL CANCER; EICOSANOIDS; GENE EXPRESSION; INFLAMMATION; LIPID METABOLISM

DOI:  https://doi.org/10.1136/gutjnl-2024-332535
J Proteome Res. 2024 Dec 09.

Development of a Multiplexed LC-MS/MS Assay for the Quantitation of Podocyte Injury Biomarkers Nephrin, Podocalyxin, and Podocin in Human Urine.

Carlos A Morales-Betanzos, Stephen P Berasi, Joel D Federspiel, Hendrik Neubert, Mireia Fernandez Ocana.

  CKD is frequently diagnosed only after a significant progression. GFR is the most common indicator of kidney function but is limited in detecting early CKD cases and distinguishing glomerular, tubular, and global CKD. Aiming to provide a glomeruli specific biomarker assay, we developed a peptide immunoaffinity targeted mass spectrometry method for the quantitation of three podocyte specific proteins in human urine: nephrin, podocalyxin, and podocin. Proteins in urine were precipitated, stable isotope labeled peptide standards incorporated, and digested with trypsin. Target peptides were enriched using an online antibody column prior to LC-MS/MS. The performance metrics for nephrin, podocalyxin, and podocin were evaluated: The lower limits of quantitation were 3.8, 22.0, and 5.4 pM, respectively. The intraplate relative error (RE) was within ±10.6%, ± 10.4%, and ±16.1%, and coefficient of variation (CV) was ≤27.2%, ≤ 14.1%, and ≤20.7% accordingly. The interplate RE was within ±7.0%, ± 3.8%, and ±3.0%, and CV was ≤17.2%, ≤ 12.1%, and ≤20.0% for the three analytes. The urinary nephrin, podocalyxin, and podocin concentrations in 60 healthy volunteers and 20 disease samples was measured, thereby establishing the basal levels of these protein and enabling future evaluation of their roles as noninvasive biomarkers of glomerular injury in the clinic.

Keywords:  CKD; LC-MS; biomarkers; glomeruli; kidney; podocytes; targeted MS

DOI:  https://doi.org/10.1021/acs.jproteome.4c00751
Bioinform Adv. 2024 ;4(1): vbae178

MeTEor: an R Shiny app for exploring longitudinal metabolomics data.

Gordon Grabert, Daniel Dehncke, Tushar More, Markus List, Anke R M Kraft, Markus Cornberg, Karsten Hiller, Tim Kacprowski.

Motivation: The availability of longitudinal omics data is increasing in metabolomics research. Viewing metabolomics data over time provides detailed insight into biological processes and fosters understanding of how systems react over time. However, the analysis of longitudinal metabolomics data poses various challenges, both in terms of statistical evaluation and visualization.
Results: To make explorative analysis of longitudinal data readily available to researchers without formal background in computer science and programming, we present MEtabolite Trajectory ExplORer (MeTEor). MeTEor is an R Shiny app providing a comprehensive set of statistical analysis methods. To demonstrate the capabilities of MeTEor, we replicated the analysis of metabolomics data from a previously published study on COVID-19 patients.
Availability and implementation: MeTEor is available as an R package and as a Docker image. Source code and instructions for setting up the app can be found on GitHub (https://github.com/scibiome/meteor). The Docker image is available at Docker Hub (https://hub.docker.com/r/gordomics/meteor). MeTEor has been tested on Microsoft Windows, Unix/Linux, and macOS.

DOI: https://doi.org/10.1093/bioadv/vbae178
Nature. 2024 Dec;636(8042): 374-379

Continuous collective analysis of chemical reactions.

Maowei Hu, Lei Yang, Nathaniel Twarog, Jason Ochoada, Yong Li, Eirinaios I Vrettos, Arnaldo X Torres-Hernandez, James B Martinez, Jiya Bhatia, Brandon M Young, Jeanine Price, Kevin McGowan, Theresa H Nguyen, Zhe Shi, Matthew Anyanwu, Mary Ashley Rimmer, Shea Mercer, Zoran Rankovic, Anang A Shelat, Daniel J Blair.

The automated synthesis of small organic molecules from modular building blocks has the potential to transform our capacity to create medicines and materials1-3. Disruptive acceleration of this molecule-building strategy broadly unlocks its functional potential and requires the integration of many new assembly chemistries. Although recent advances in high-throughput chemistry4-6 can speed up the development of appropriate synthetic methods, for example, in selecting appropriate chemical reaction conditions from the vast range of potential options, equivalent high-throughput analytical methods are needed. Here we report a streamlined approach for the rapid, quantitative analysis of chemical reactions by mass spectrometry. The intrinsic fragmentation features of chemical building blocks generalize the analyses of chemical reactions, allowing sub-second readouts of reaction outcomes. Central to this advance was identifying that starting material fragmentation patterns function as universal barcodes for downstream product analysis by mass spectrometry. Combining these features with acoustic droplet ejection mass spectrometry7,8 we could eliminate slow chromatographic steps and continuously evaluate chemical reactions in multiplexed formats. This enabled the assignment of reaction conditions to molecules derived from ultrahigh-throughput chemical synthesis experiments. More generally, these results indicate that fragmentation features inherent to chemical synthesis can empower rapid data-rich experimentation.

DOI: https://doi.org/10.1038/s41586-024-08211-4
Cell Rep. 2024 Dec 12. pii: S2211-1247(24)01357-3. [Epub ahead of print]43(12): 115006

Carnitine palmitoyltransferase 1 facilitates fatty acid oxidation in a non-cell-autonomous manner.

Joseph Choi, Danielle M Smith, Susanna Scafidi, Ryan C Riddle, Michael J Wolfgang.

  Mitochondrial fatty acid oxidation is facilitated by the combined activities of carnitine palmitoyltransferase 1 (Cpt1) and Cpt2, which generate and utilize acylcarnitines, respectively. We compare the response of mice with liver-specific deficiencies in the liver-enriched Cpt1a or the ubiquitously expressed Cpt2 and discover that they display unique metabolic, physiological, and molecular phenotypes. The loss of Cpt1a or Cpt2 results in the induction of the muscle-enriched isoenzyme Cpt1b in hepatocytes in a Pparα-dependent manner. However, hepatic Cpt1b does not contribute substantively to hepatic fatty acid oxidation when Cpt1a is absent. Liver-specific double knockout of Cpt1a and Cpt1b or Cpt2 eliminates the mitochondrial oxidation of non-esterified fatty acids. However, Cpt1a/Cpt1b double knockout mice retain fatty acid oxidation by utilizing extracellular long-chain acylcarnitines that are dependent on Cpt2. These data demonstrate the non-cell-autonomous intercellular metabolism of fatty acids in hepatocytes.

Keywords:  CP: Metabolism; Cpt1; Cpt2; acylcarnitine; biochemistry; fasting; liver; metabolism

DOI:  https://doi.org/10.1016/j.celrep.2024.115006
J Bone Miner Res. 2024 Dec 08. pii: zjae195. [Epub ahead of print]

Enhanced fatty acid oxidation in osteoprogenitor cells provides protection from high-fat diet induced bone dysfunction.

Ananya Nandy, Ron C M Helderman, Santosh Thapa, Sun H Peck, Alison Richards, Shobana Jayapalan, Nikita Narayani, Michael P Czech, Clifford J Rosen, Elizabeth Rendina-Ruedy.

  Bone homeostasis within the skeletal system is predominantly maintained by bone formation and resorption. Where formation of new bone involves maturation of stromal cells to mineral and matrix secreting mature osteoblasts, which requires cellular energy or adenosine triphosphate (ATP). Alterations in systemic metabolism can influence osteoblast functioning. In line with this, type 2 diabetes mellitus (T2DM), a common metabolic disorder is also associated with reduced bone formation and increased risk of fracture. Impairment in lipid metabolism is one of the key features associated with T2DM-related pathologies in multiple tissues. Therefore, we tested the hypothesis that the reduced bone formation reported in obese murine models of impaired glucose tolerance is a function of disrupted lipid metabolism in osteoblasts. We first confirmed that mice fed a high fat diet have reduced bone microarchitecture along with lower bone formation rates. Interestingly, osteoblasts from obese mice harbor higher numbers of cytosolic lipid droplets along with decreased bioenergetic profiles compared to control cells. Further supporting this observation, bone tibia cortex demonstrated higher total lipid content in high fat diet fed mice compared to control-fed mice. As a further proof of principle, we generated a novel murine model to conditionally delete Plin2 in osteoblast-progenitor cells using Prrx1-Cre, to enhance lipid droplet breakdown. Our data demonstrate that knocking down Plin2 in an osteoprogenitor specific manner protects from high-fat diet induced osteoblast dysfunction. Furthermore, the mechanism of action involves enhanced osteoblast fatty acid oxidation. In conclusion, the current studies establish that high fat diet induced glucose intolerance leads to perturbations in osteoblast lipid metabolism, thus causing lower bone formation, which can be protected against by increasing fatty acid oxidation.

Keywords:  diabetes; fracture; lipids; metabolism; perilipin 2

DOI:  https://doi.org/10.1093/jbmr/zjae195