bims-mascan 2025-03-16 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2025–03–16
seventeen papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

Automated Sequential Derivatization for Gas Chromatography-[Orbitrap] Mass Spectrometry-based Metabolite Profiling of Human Blood-based Samples.
In-depth plasma N-glycoproteome profiling using narrow-window data-independent acquisition on the Orbitrap Astral mass spectrometer.
Enhanced lipidomics workflows for plasma and extracellular vesicles through advanced liquid chromatography-tandem mass spectrometry integrated.
dELTA-MS: A Mass Spectrometry-Based Proteomics Approach for Identifying ADP-Ribosylation Sites and Forms.
Application of Metabolomics in Carcinogenesis and Cancer Prevention by Dietary Phytochemicals.
A Review of Retention Time Alignment Methods and Tools for Proteomics Data.
Deciphering Colorectal Cancer-Hepatocyte Interactions: A Multiomics Platform for Interrogation of Metabolic Crosstalk in the Liver-Tumor Microenvironment.
Rapid Proteomic Amelogenin Sex Estimation of Human and Cattle Remains Using Untargeted Evosep-timsTOF Mass Spectrometry.
Glucose Metabolism and Tumor Microenvironment: Mechanistic Insights and Therapeutic Implications.
Quantitative method for intestinal short chain fatty acids based on stable isotope labeling combined with liquid chromatography-mass spectrometry.
Tear Sampling and Biomarker Discovery: A Robust Workflow for Routine Clinical Applications Using UHPLC-MS/MS and Schirmer Strips.
Plasma Lipidomics of Preadolescent Children: A Hokkaido Study.
Development of an Adaptive, Economical, and Easy-to-Use SP3-TMT Automated Sample Preparation Workflow for Quantitative Proteomics.
Islet hormones at the intersection of glucose and amino acid metabolism.
Succinate dehydrogenase activity supports de novo purine synthesis.
Maximizing Immunopeptidomics-Based Bacterial Epitope Discovery by Multiple Search Engines and Rescoring.
Classification of Collagens via Peptide Ambiguation, in a Paleoproteomic LC-MS/MS-Based Taxonomic Pipeline.

Bio Protoc. 2025 Mar 05. 15(5): e5196

Automated Sequential Derivatization for Gas Chromatography-[Orbitrap] Mass Spectrometry-based Metabolite Profiling of Human Blood-based Samples.

Akrem Jbebli, Kateřina Coufalíková, Moira Zanaboni, Manuela Bergna, Renzo Picenoni, Jana Klánová, Elliott J Price.

  Many small molecules require derivatization to increase their volatility and to be amenable to gas chromatographic (GC) separation. Derivatization is usually time-consuming, and typical batch-wise procedures increase sample variability. Sequential automation of derivatization via robotic liquid handling enables the overlapping of sample preparation and analysis, maximizing time efficiency and minimizing variability. Herein, a protocol for the fully automated, two-stage derivatization of human blood-based samples in line with GC-[Orbitrap] mass spectrometry (MS)-based metabolomics is described. The protocol delivers a sample-to-sample runtime of 31 min, being suitable for better throughput routine metabolomic analysis. Key features • Direct and rapid methoximation on vial followed by silylation of metabolites in various blood matrices. • Measure ~40 samples per 24 h, identifying > 70 metabolites. • Quantitative reproducibility of routinely measured metabolites with coefficients of variation (CVs) < 30%. • Requires a Thermo ScientificTM TriPlusTM RSH (or comparable) autosampler equipped with incubator/agitator, cooled drawer, and automatic tool change (ATC) station equipped with liquid handling tools. Graphical overview Workflow for profiling metabolites in human blood using automated derivatization.

Keywords:  Automation; Derivatization; Gas chromatography–mass spectrometry (GC–MS); Metabolite profiling; Thermo ScientificTM TriPlusTM RSH

DOI:  https://doi.org/10.21769/BioProtoc.5196
Nat Commun. 2025 Mar 13. 16(1): 2497

In-depth plasma N-glycoproteome profiling using narrow-window data-independent acquisition on the Orbitrap Astral mass spectrometer.

Shelley Jager, Martin Zeller, Anna Pashkova, Douwe Schulte, Eugen Damoc, Karli R Reiding, Alexander A Makarov, Albert J R Heck.

Recently, a conceptually new mass analyzer was introduced by pairing a quadrupole Orbitrap mass spectrometer with an asymmetric track lossless (Astral™) analyzer. This system provides >200 Hz MS/MS scanning speed, high resolving power, sensitivity, and mass accuracy. Due to its speed, the instrument allows for a narrow-window data-independent acquisition (nDIA) strategy, representing a new technical milestone in peptide-centric proteomics. However, this new system may also be applied to other complex and clinically important proteomes, such as the human plasma N-glycoproteome. Here, we evaluate the Orbitrap Astral mass spectrometer for the in-depth analysis of the plasma N-glycoproteome and pioneer a dedicated nDIA workflow, termed "nGlycoDIA", on glycopeptide enriched and crude plasma. This strategy leads to the cumulative identification of over 3000 unique glycoPSMs derived from 181 glycoproteins in just 40 minutes and covers a dynamic range of 7 orders of magnitude for a glycopeptide enriched plasma sample. Notably, we detect several glycosylated cytokines that have reported plasma concentrations in the ng/L range. Furthermore, shortening the gradient to 10 min still allows for the detection of almost 1850 (95% CI [1840-1860]) unique glycoPSMs, indicating that high-throughput in-depth clinical plasma glycoproteomics may be within reach.

DOI: https://doi.org/10.1038/s41467-025-57916-1
Talanta. 2025 Mar 01. pii: S0039-9140(25)00337-6. [Epub ahead of print]291 127847

Enhanced lipidomics workflows for plasma and extracellular vesicles through advanced liquid chromatography-tandem mass spectrometry integrated.

Adriana F L Vilela, Miguel R Patrício, Pedro Nobre-Azevedo, Jonatan C S de Carvalho, Thiago V Defelippo-Felippe, Nathan N H Pontes, Daniel L Rodrigues, Bianca T M Oliveira, Pedro V da Silva-Neto, Viviani Nardini, Ana P M Fernandes, Fausto Almeida, Lucia H Faccioli, Carlos A Sorgi.

  Lipidomics, a subfield of metabolomics, provides comprehensive analysis of lipids in biological systems and is essential for biomedical research, driven by advances in analytical technologies. Lipids are crucial biomolecules in cellular functions and have been increasingly recognized for their role in physiological and pathological processes. This study focuses on advanced strategies for the development, validation, and implementation of untargeted lipidomics methods in human plasma and extracellular vesicles (EVs) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Method validation demonstrated excellent accuracy (precision and trueness) (81-120 % of nominal value), precision with inter-day repeatability below 20 %, limits of quantification ranging from 0.25 to 25 μM, and recovery rates exceeding 80 % for most lipid classes, as well as matrix effects. Plasma samples were used as a proof-of-concept study, and the method was ultimately applied to human macrophage-derived EVs. Lipid extraction utilized four liquid-liquid extraction methods to ensure broad lipid class coverage, high recovery, and repeatability. Additionally, we demonstrated that a sonication-assisted homogenization step effectively facilitates lipid extraction from EVs. Through untargeted lipidomics, our study identifies and quantifies a diverse range of lipid species in human plasma (225 lipids analytes) and macrophage-derived EVs (124 lipids analytes) within different classes. Overall, we present sophisticated approaches that combine pre-analytical lipid extraction techniques with high-resolution LC-MS/MS to enhance lipidomics research. This approach enhances the characterization of lipid profiles and their biological implications, paving the way for applications in personalized medicine and the discovery of novel lipid biomarkers associated with EVs biogenesis.

Keywords:  Extracellular vesicles; LC-MS/MS; Lipid-extraction; Lipidomics

DOI:  https://doi.org/10.1016/j.talanta.2025.127847
J Proteome Res. 2025 Mar 13.

dELTA-MS: A Mass Spectrometry-Based Proteomics Approach for Identifying ADP-Ribosylation Sites and Forms.

Isabel R Uribe, Emily Zahn, Richard Searfoss, Han-Byeol Kim, Morgan Dasovich, Jim Voorneveld, Sabrina R Hunt, Ugochi C Onuoha, Catherine Valadez, Dmitri V Filippov, Chan Hyun Na, Benjamin A Garcia, Benjamin C Orsburn, Anthony K L Leung.

  ADP-ribosylation, characterized by the addition of adenosine diphosphate ribose, can occur in both monomeric (MARylation) and polymeric (PARylation) forms. Little is known about the specific contributions of MARylation and PARylation to cellular processes due to a lack of tools for jointly investigating these individual forms. We present a novel mass spectrometry (MS)-based proteomics approach that preserves information about the native ADP-ribosylation form associated with the modification site within a single proteomics experiment. Our workflow enables the simplified, binary identification of ADP-ribosylation forms, avoiding some challenges typically presented by PARylated peptides during MS analysis. Our method uses the coronaviral enzyme NS2 to reverse our previous labeling approach, ELTA, which enzymatically labels the terminal ADP-ribose. NS2 deconjugates ELTA-labeled free or peptide-conjugated ADP-ribose monomers and polymers (thereby termed dELTA), leaving behind a signature phosphate. Our dELTA-MS workflow involves ELTA labeling, dELTA deconjugation, and further processing using Deinococcus radiodurans poly(ADP-ribose) glycohydrolase (DrPARG), resulting in two distinct mass shifts for MARylation and PARylation sites. We demonstrate the feasibility of this workflow for proteomics analyses using proof-of-principle peptide standards. dELTA-MS thus creates possibilities to reveal the fundamental biology of ADP-ribosylation and explore its dysregulation, in terms of both sites and forms, associated with disease progression.

Keywords:  ADP-ribosylation; PARPs; mono-ADP-ribosylation; poly-ADP-ribosylation; post-translational modification; proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.4c00890
Curr Pharmacol Rep. 2025 ;11(1): 12

Application of Metabolomics in Carcinogenesis and Cancer Prevention by Dietary Phytochemicals.

Rebecca Mary Peter, Xiaoyang Su, Ah-Ng Kong.

   Purpose of Review: In this review article, specific emphasis is on evolution of metabolomics in cancer research, metabolomics workflow, general understanding of liquid chromatography - mass spectrometry (LC-MS) based platform for quantitation of metabolites, their biological interpretation and the application in carcinogenesis and cancer prevention by dietary phytochemicals.
Recent Findings: Metabolomics is increasingly becoming a preferred approach for next generation metabolic screening and has profound impact on medical practice. Metabolomics describes the end products of biochemical processes which are greatly influenced by genetic and environmental factors. Metabolic alterations can be linked to potential biochemical reactions/enzymes and their corresponding genes. Thus, these results can be further validated via multi-omics approach including genomics, transcriptomics and proteomics. However, challenges exist within and between omic-domain data integration considering complex biochemical regulation including organism versus tissue versus cellular level processes, epigenetics, transcriptional and post translational modifications. Metabolomics can reflect the steady state or dynamic state of metabolism because metabolites are highly dynamic in space and time.
Summary: Metabolomic analysis of biological samples exhibit the possibility to determine mechanism of action of anti-cancer agents, biomarker discovery and impact of genetic alterations.

Keywords:  Cancer prevention; Liquid chromatography – mass spectrometry; Metabolic alterations; Metabolomic workflow; Metabolomics; Phytochemicals

DOI:  https://doi.org/10.1007/s40495-025-00396-0
Proteomics. 2025 Mar 09. e202400088

A Review of Retention Time Alignment Methods and Tools for Proteomics Data.

Yi Liu, Kaikun Xu, Cheng Chang.

  Mass spectrometry (MS) is widely used in proteomics research in recent years. In practice, MS is often coupled with liquid chromatography or gas chromatography to analyze complex mixtures. Retention time (RT) is a critical feature that distinguishes different components in mixtures. And RT alignment is one of the crucial methods for correlating identical components across different samples. This review provides a broad perspective of RT alignment methods and tools used in proteomics and metabolomics. Firstly, we categorize these methods and tools into four types based on the information they employ, and introduce them, respectively. Subsequently, we present the application of RT alignment in proteomics. We have emphasized that the identification-free strategy might be a potential application of RT alignment. Finally, we summarize the limitations of current methods and discuss the future direction of RT alignment.

Keywords:  bioinformatics; mass spectrometry; proteomics; retention time alignment

DOI:  https://doi.org/10.1002/pmic.202400088
Int J Mol Sci. 2025 Feb 25. pii: 1976. [Epub ahead of print]26(5):

Deciphering Colorectal Cancer-Hepatocyte Interactions: A Multiomics Platform for Interrogation of Metabolic Crosstalk in the Liver-Tumor Microenvironment.

Alisa B Nelson, Lyndsay E Reese, Elizabeth Rono, Eric D Queathem, Yinjie Qiu, Braedan M McCluskey, Alexandra Crampton, Eric Conniff, Katherine Cummins, Ella Boytim, Senali Dansou, Justin Hwang, Sandra E Safo, Patrycja Puchalska, David K Wood, Kathryn L Schwertfeger, Peter A Crawford.

  Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to adapt to and exploit their microenvironment for sustained growth. The liver is a common site of metastasis, but the interactions between tumor cells and hepatocytes remain poorly understood. In the context of liver metastasis, these interactions play a crucial role in promoting tumor survival and progression. This study leverages multiomics coverage of the microenvironment via liquid chromatography and high-resolution, high-mass-accuracy mass spectrometry-based untargeted metabolomics, 13C-stable isotope tracing, and RNA sequencing to uncover the metabolic impact of co-localized primary hepatocytes and a colon adenocarcinoma cell line, SW480, using a 2D co-culture model. Metabolic profiling revealed disrupted Warburg metabolism with an 80% decrease in glucose consumption and 94% decrease in lactate production by hepatocyte-SW480 co-cultures relative to SW480 control cultures. Decreased glucose consumption was coupled with alterations in glutamine and ketone body metabolism, suggesting a possible fuel switch upon co-culturing. Further, integrated multiomics analysis indicates that disruptions in metabolic pathways, including nucleoside biosynthesis, amino acids, and TCA cycle, correlate with altered SW480 transcriptional profiles and highlight the importance of redox homeostasis in tumor adaptation. Finally, these findings were replicated in three-dimensional microtissue organoids. Taken together, these studies support a bioinformatic approach to study metabolic crosstalk and discovery of potential therapeutic targets in preclinical models of the tumor microenvironment.

Keywords:  cancer metabolism; metabolomics; multiomics; tumor microenvironment

DOI:  https://doi.org/10.3390/ijms26051976
Rapid Commun Mass Spectrom. 2025 Apr 15. 39(11): e10022

Rapid Proteomic Amelogenin Sex Estimation of Human and Cattle Remains Using Untargeted Evosep-timsTOF Mass Spectrometry.

Charllotte Blacka, Adam Dowle, Mik Lisowski, Michelle Alexander, Jessica Hendy, Kirsty Penkman, Jackie Mosely.

   RATIONALE: Sex estimation by analysis of amelogenin peptides in archaeological and fossil material has recently been gaining great traction within the fields of archaeology and palaeontology. Current widely used proteomic amelogenin sex estimation methods are hindered by relatively long mass spectrometric run times, or targeting peptides specific to human amelogenin proteins. Untargeted, high-throughput amelogenin sexing would be invaluable for a range of applications, from sex estimation of remains at mass grave sites to broadening the application of rapid amelogenin sexing to non-hominin species for husbandry and evolutionary studies.
METHODS: A new acid etch protocol followed by Evosep-LC-TIMS-TOF mass spectrometry is presented for amelogenin analysis, providing global peptide data through rapid mass spectrometric methods in under 20 min per sample (including sample preparation, mass spectrometric acquisition and data processing). This sampling protocol was developed on modern cattle (Bos taurus) teeth, before Evosep-timsTOF partial validation with archaeological cattle and human (Homo sapiens) teeth, demonstrating the potential of straightforward application of this rapid amelogenin sexing method to a range of taxa.
RESULTS: The rapid Evosep-LC-TIMS-TOF mass spectrometry methods gave comparable peptide counts to conventional long untargeted methods, while maintaining similar (or faster) acquisition times to those reported in methods targeting specific human amelogenin peptides. Implementation of the novel acid etch sampling approach also streamlined sample preparation without compromising peptide counts.
CONCLUSIONS: Rapid, untargeted Evosep-LC-TIMS-TOF mass spectrometry was successfully implemented in sex estimation of modern and archaeological material from Bos taurus and Homo sapiens teeth. This demonstrates an advancement in low-cost, high-throughput amelogenin sex estimation, for both human and zooarchaeological applications.

Keywords:  LC–MS/MS; TIMS‐TOF; amelogenin; palaeoproteomics; sex estimation

DOI:  https://doi.org/10.1002/rcm.10022
Int J Mol Sci. 2025 Feb 22. pii: 1879. [Epub ahead of print]26(5):

Glucose Metabolism and Tumor Microenvironment: Mechanistic Insights and Therapeutic Implications.

Wiktoria Andryszkiewicz, Julia Gąsiorowska, Maja Kübler, Karolina Kublińska, Agata Pałkiewicz, Adam Wiatkowski, Urszula Szwedowicz, Anna Choromańska.

  Metabolic reprogramming in cancer cells involves changes in glucose metabolism, glutamine utilization, and lipid production, as well as promoting increased cell proliferation, survival, and immune resistance by altering the tumor microenvironment. Our study analyzes metabolic reprogramming in neoplastically transformed cells, focusing on changes in glucose metabolism, glutaminolysis, and lipid synthesis. Moreover, we discuss the therapeutic potential of targeting cancer metabolism, focusing on key enzymes involved in glycolysis, the pentose phosphate pathway, and amino acid metabolism, including lactate dehydrogenase A, hexokinase, phosphofructokinase and others. The review also highlights challenges such as metabolic heterogeneity, adaptability, and the need for personalized therapies to overcome resistance and minimize adverse effects in cancer treatment. This review underscores the significance of comprehending metabolic reprogramming in cancer cells to engineer targeted therapies, personalize treatment methodologies, and surmount challenges, including metabolic plasticity and therapeutic resistance.

Keywords:  cancer stem cells; glycolysis; hexokinase; lactate dehydrogenase A; phosphofructokinase; pyruvate kinase; the Warburg effect; tumor microenvironment; tumor-associated macrophages

DOI:  https://doi.org/10.3390/ijms26051879
J Pharm Biomed Anal. 2025 Mar 10. pii: S0731-7085(25)00139-6. [Epub ahead of print]260 116798

Quantitative method for intestinal short chain fatty acids based on stable isotope labeling combined with liquid chromatography-mass spectrometry.

Yang Zhang, Yan Qiao, Xin Ding, Ya Zhang, Xuemei Su, Lei Zhang, Qian Zhou, Guangguo Tan.

  Short chain fatty acids (SCFAs) are produced from the breakdown of dietary proteins and fiber by gut microbes, and they have a close relationship with the health and diseases of the host. However, due to the similar structures of SCFAs, the abundance of active molecules, the wide concentration range in biological samples, and the characteristics such as high polarity, poor chromatographic separation, and ionization performance, it is challenging to comprehensively and accurately quantify SCFAs. This study utilized a stable isotope-labeled carboxyl derivatization reagent d0-/d6-2,4-dimethoxy-6-piperazin-1-yl pyrimidine (d0-/d6-DMPP) to establish a new method for the wide-coverage quantification analysis of SCFAs using ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS), capable of detecting the content of 16 SCFAs. The method demonstrated low limits of detection (LODs) of 0.05-0.5 nmol/L and limits of quantification (LOQs) of 0.1-1.0 nmol/L, with excellent linearity (R² > 0.99), intra-day precision (RSD < 8.5 %), and inter-day precision (RSD < 7.8 %). Using this quantitative analysis method, we successfully quantified 16 SCFAs from the colonic contents of rats with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ulcerative colitis (UC) and Sini decoction (SND) intervention. It was found that after the interventional treatment with SND, the levels of 7 SCFAs in the colonic contents of rats with UC were significantly up regulated, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, 2-methylbutyric acid, and hexanoic acid, while 4 SCFAs were significantly down regulated, including 3-hydroxyisovaleric, 3-methyl-2-oxobutanoic acid, 3-methyl-2-oxovaleric acid, and 4-methyl-2-oxovaleric acid. These findings suggested that SND might exert its therapeutic effect on UC by regulating the metabolism of SCFAs. Overall, this study not only provides a new method for the analysis of SCFAs with high sensitivity and wide-coverage but also offers important scientific evidence for understanding the mechanism of SND against UC.

Keywords:  Derivatization; Quantification; Short chain fatty acids; Sini decoction; Stable isotope labeling; Ulcerative colitis

DOI:  https://doi.org/10.1016/j.jpba.2025.116798
Int J Mol Sci. 2025 Feb 26. pii: 2041. [Epub ahead of print]26(5):

Tear Sampling and Biomarker Discovery: A Robust Workflow for Routine Clinical Applications Using UHPLC-MS/MS and Schirmer Strips.

Rossana Comito, Carmen Ciavarella, Gloria Astolfi, Matteo Conti, Emanuele Porru, Francesco Saverio Violante, Piera Versura.

  Human tear analysis is gaining increasing attention as a non-invasive tool for several applications such as proteomics and biomarker identification in various diseases, including cancer. The choice of the correct sampling method determines the result of the analysis. In this study, we developed and validated a robust method for tear protein quantification using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Tear samples were collected with Schirmer strips, a low-cost and practical tool for tear collection. It is the first time that internal standards have been used to enhance the analytical performance of a method based on Schirmer strips for tear sampling, overcoming the issues widely reported in the literature regarding protein extraction and data reproducibility. Non-human proteins were used for method development, ensuring improved accuracy and analytical precision. The method demonstrated excellent recovery, high sensitivity, and reproducibility. The use of Schirmer strips, combined with this advanced analytical method, highlights their potential as a reliable support for tear protein quantification and biomarker discovery. This study provides a cost-effective and reliable workflow for tear proteome analysis and contributes to the growing field of tear-based diagnostics, making it suitable for routine clinical and research applications in precision medicine.

Keywords:  Schirmer strip; cancer biomarkers; mass spectrometry; non-invasive matrices; proteomics; tear film

DOI:  https://doi.org/10.3390/ijms26052041
J Lipids. 2025 ;2025 3106145

Plasma Lipidomics of Preadolescent Children: A Hokkaido Study.

Jayashankar Jayaprakash, Siddabasave Gowda B Gowda, Divyavani Gowda, Atsuko Ikeda, Yu Ait Bamai, Rahel Mesfin Ketema, Reiko Kishi, Yifan Chen, Hitoshi Chiba, Shu-Ping Hui.

  Lipids are the most abundant biomolecules of human plasma, and their balance plays a significant role in health and disease management. Despite the importance of lipids, the studies focused on the comprehensive determination of the plasma lipidome in children are limited. In this study, we investigated the sex, age, and weight-specific changes in the plasma lipidome of nonfasting preadolescent children aged 9-12 years (n = 342) using a nontargeted liquid chromatography-mass spectrometry technique. A total of 219 lipid species were characterized in the plasma samples. Multivariate analysis revealed that boys and girls have similar lipid profiles, but relatively higher levels of capric acid-composed triacylglycerols (TGs) were observed in plasma samples of boys. Saturated fatty acids are the most abundant fatty acyls followed by mono- and polyunsaturated fatty acids in the plasma of both boys and girls. Sphingolipids such as ceramides, hexosylceramides, sphingomyelin, and a phospholipid (phosphatidylinositol) were relatively higher in the plasma of a 10-year-old group than other age groups. Plasma levels of TG and phosphatidylserine were increased within age from 9 to 12 years. Furthermore, most of the TG molecular species were increased in the plasma of overweight children compared to the normal range groups. The receiver operating characteristic analysis results show that TG (10:0/10:0/18:1) could be a specific marker for childhood obesity (area under the curve (AUC) = 0.72). Overall, this study highlights the altered plasma lipidome in preadolescent children for sex, age, and percentage of overweight. Early detection of lipid markers for obesity would be a promising target for developing therapeutic strategies.

Keywords:  childhood obesity; children; lipidomics; liquid chromatography; mass spectrometry; plasma

DOI:  https://doi.org/10.1155/jl/3106145
bioRxiv. 2025 Feb 27. pii: 2025.02.23.639731. [Epub ahead of print]

Development of an Adaptive, Economical, and Easy-to-Use SP3-TMT Automated Sample Preparation Workflow for Quantitative Proteomics.

Jake N Hermanson, Lea A Barny, Lars Plate.

Liquid handling robots have been developed to automate various steps of the bottom-up proteomics workflow, however, protocols for the generation of isobarically labeled peptides remain limited. Existing methods often require costly specialty devices and are constrained by fixed workflows. To address this, we developed a cost-effective, flexible, automated sample preparation protocol for TMT-labeled peptides using the Biomek i5 liquid handler. Our approach leverages Single-Pot Solid-Phase-Enhanced Sample Preparation (SP3) with paramagnetic beads to streamline protein cleanup and digestion. The protocol also allows for adjustment of trypsin concentration and peptide-to-TMT ratio to increase throughput and reduce costs, respectively. We compared our automated and manual 18-plex TMT-Pro labeling workflows by monitoring select protein markers of the Unfolded Protein Response (UPR) in pharmacologically activatable, engineered cell lines. Overall, the automated protocol demonstrated equivalent performance in peptide and protein identifications, digestion and labeling efficiency, and an enhancement in the dynamic range of TMT quantifications. Compared to the manual method, the Biomek protocol significantly reduces hands-on time and minimizes sample handling errors. The 96-well format additionally allows for the number of TMT reactions to be scaled up quickly without a significant increase in user interaction. Our optimized automated workflow enhances throughput, reproducibility, and cost-effectiveness, making it a valuable tool for high-throughput proteomics studies.

DOI: https://doi.org/10.1101/2025.02.23.639731
Nat Rev Endocrinol. 2025 Mar 07.

Islet hormones at the intersection of glucose and amino acid metabolism.

Phillip J White, Nicolai J Wewer Albrechtsen, Jonathan E Campbell.

The pancreatic islets of Langerhans are central to fine-tuning metabolism to ensure metabolic homeostasis during the transition between fasting and feeding. Insulin and glucagon, the principal hormones generated and secreted by islets, exert powerful control in various metabolic tissues to drive nutrient uptake, storage and metabolism. Their canonical actions on glycaemia have positioned these hormones in opposition, however, their metabolic actions extend beyond controlling blood levels of glucose. Indeed, these islet hormones are just as influential in regulating lipid and amino acid metabolism and it is becoming clear that many of these actions involve an interplay between insulin and glucagon, which is contrary to the dogmatic view that these hormones are antagonistic in nature. Finally, we postulate that examining the effects of islet hormones on the metabolism of individual metabolites is overly simplistic. Here, we discuss the actions of each islet hormone alone and in combination with the others in regulating glucose and amino acid metabolism and explore how these signalling networks are closely linked and strongly influence one another.

DOI: https://doi.org/10.1038/s41574-025-01100-4
bioRxiv. 2025 Mar 01. pii: 2025.02.26.640389. [Epub ahead of print]

Succinate dehydrogenase activity supports de novo purine synthesis.

Mushtaq A Nengroo, Austin T Klein, Heather S Carr, Olivia Vidal-Cruchez, Umakant Sahu, Daniel J McGrail, Nidhi Sahni, Peng Gao, John M Asara, Hardik Shah, Marc L Mendillo, Issam Ben-Sahra.

The de novo purine synthesis pathway is fundamental for nucleic acid production and cellular energetics, yet the role of mitochondrial metabolism in modulating this process remains underexplored. In many cancers, metabolic reprogramming supports rapid proliferation and survival, but the specific contributions of the tricarboxylic acid (TCA) cycle enzymes to nucleotide biosynthesis are not fully understood. Here, we demonstrate that the TCA cycle enzyme succinate dehydrogenase (SDH) is essential for maintaining optimal de novo purine synthesis in normal and cancer cells. Genetic or pharmacological inhibition of SDH markedly attenuates purine synthesis, leading to a significant reduction in cell proliferation. Mechanistically, SDH inhibition causes an accumulation of succinate, which directly impairs the purine biosynthetic pathway. In response, cancer cells compensate by upregulating the purine salvage pathway, a metabolic adaptation that represents a potential therapeutic vulnerability. Notably, co-inhibition of SDH and the purine salvage pathway induces pronounced antiproliferative and antitumoral effects in preclinical models. These findings not only reveal a signaling role for mitochondrial succinate in regulating nucleotide metabolism but also provide a promising therapeutic strategy for targeting metabolic dependencies in cancer.

DOI: https://doi.org/10.1101/2025.02.26.640389
J Proteome Res. 2025 Mar 13.

Maximizing Immunopeptidomics-Based Bacterial Epitope Discovery by Multiple Search Engines and Rescoring.

Patrick Willems, Fabien Thery, Laura Van Moortel, Margaux De Meyer, An Staes, Adillah Gul, Lyudmila Kovalchuke, Arthur Declercq, Robbe Devreese, Robbin Bouwmeester, Ralf Gabriels, Lennart Martens, Francis Impens.

  Mass spectrometry-based discovery of bacterial immunopeptides presented by infected cells allows untargeted discovery of bacterial antigens that can serve as vaccine candidates. However, reliable identification of bacterial epitopes is challenged by their extremely low abundance. Here, we describe an optimized bioinformatic framework to enhance the confident identification of bacterial immunopeptides. Immunopeptidomics data of cell cultures infected with Listeria monocytogenes were searched by four different search engines, PEAKS, Comet, Sage and MSFragger, followed by data-driven rescoring with MS2Rescore. Compared with individual search engine results, this integrated workflow boosted immunopeptide identification by an average of 27% and led to the high-confidence detection of 18 additional bacterial peptides (+27%) matching 15 different Listeria proteins (+36%). Despite the strong agreement between the search engines, a small number of spectra (<1%) had ambiguous matches to multiple peptides and were excluded to ensure high-confidence identifications. Finally, we demonstrate our workflow with sensitive timsTOF SCP data acquisition and find that rescoring, now with inclusion of ion mobility features, identifies 76% more peptides compared to Q Exactive HF acquisition. Together, our results demonstrate how integration of multiple search engine results along with data-driven rescoring maximizes immunopeptide identification, boosting the detection of high-confidence bacterial epitopes for vaccine development.

Keywords:  Listeria monocytogenes; TIMS2Rescore; immunopeptides; ion mobility; mass spectrometry; search engines

DOI:  https://doi.org/10.1021/acs.jproteome.4c00864
J Proteome Res. 2025 Mar 13.

Classification of Collagens via Peptide Ambiguation, in a Paleoproteomic LC-MS/MS-Based Taxonomic Pipeline.

Ian Engels, Alexandra Burnett, Prudence Robert, Camille Pironneau, Grégory Abrams, Robbin Bouwmeester, Peter Van der Plaetsen, Kévin Di Modica, Marcel Otte, Lawrence Guy Straus, Valentin Fischer, Fabrice Bray, Bart Mesuere, Isabelle De Groote, Dieter Deforce, Simon Daled, Maarten Dhaenens.

  Liquid chromatography-mass spectrometry (LC-MS/MS) extends the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) Zooarcheology by Mass Spectrometry (ZooMS) "mass fingerprinting" approach to species identification by providing fragmentation spectra for each peptide. However, ancient bone samples generate sparse data containing only a few collagen proteins, rendering target-decoy strategies unusable and increasing uncertainty in peptide annotation. To ameliorate this issue, we present a ZooMS/MS data pipeline that builds on a manually curated Collagen database and comprises two novel algorithms: isoBLAST and ClassiCOL. isoBLAST first extends peptide ambiguity by generating all "potential peptide candidates" isobaric to the annotated precursor. The exhaustive set of candidates created is then used to retain or reject different potential paths at each taxonomic branching point from superkingdom to species, until the greatest possible specificity is reached. Uniquely, ClassiCOL allows for the identification of taxonomic mixtures, including contaminated samples, as well as suggesting taxonomies not represented in sequence databases, including extinct taxa. All considered ambiguity is then graphically represented with clear prioritization of the potential taxa in the sample. Using public as well as in-house data acquired on different instruments, we demonstrate the performance of this universal postprocessing and explore the identification of both genetic and sample mixtures. Diet reconstruction from 40,000-year-old cave hyena coprolites illustrates the exciting potential of this approach.

Keywords:  Belgium; ClassiCOL; ZooMS; ZooMS/MS; archeology; bioinformatics; isoBLAST; mass spectrometry; paleoproteomics; proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.4c00962