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Proteomes, Volume 13, Issue 1 (March 2025) – 9 articles

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25 pages, 3722 KiB  
Article
Systems Biology of Recombinant 2G12 and 353/11 mAb Production in CHO-K1 Cell Lines at Phosphoproteome Level
by Eldi Sulaj, Felix L. Sandell, Linda Schwaigerlehner, Gorji Marzban, Juliane C. Dohm and Renate Kunert
Proteomes 2025, 13(1), 9; https://doi.org/10.3390/proteomes13010009 (registering DOI) - 10 Feb 2025
Viewed by 125
Abstract
Background: Chinese hamster ovary (CHO) cells are extensively used in the pharmaceutical industry for producing complex proteins, primarily because of their ability to perform human-like post-translational modifications. However, the efficiency of high-quality protein production can vary significantly for monoclonal antibody-producing cell lines, [...] Read more.
Background: Chinese hamster ovary (CHO) cells are extensively used in the pharmaceutical industry for producing complex proteins, primarily because of their ability to perform human-like post-translational modifications. However, the efficiency of high-quality protein production can vary significantly for monoclonal antibody-producing cell lines, within the CHO host cell lines or by extrinsic factors. Methods: To investigate the complex cellular mechanisms underlying this variability, a phosphoproteomics analysis was performed using label-free quantitative liquid chromatography after a phosphopeptide enrichment of recombinant CHO cells producing two different antibodies and a tunicamycin treatment experiment. Using MaxQuant and Perseus for data analysis, we identified 2109 proteins and quantified 4059 phosphosites. Results: Significant phosphorylation dynamics were observed in nuclear proteins of cells producing the difficult-to-produce 2G12 mAb. It suggests that the expression of 2G12 regulates nuclear pathways based on increases and decreases in phosphorylation abundance. Furthermore, a substantial number of changes in the phosphorylation pattern related to tunicamycin treatment have been detected. TM treatment affects, among other phosphoproteins, the eukaryotic elongation factor 2 kinase (Eef2k). Conclusions: The alterations in the phosphorylation landscape of key proteins involved in cellular processes highlight the mechanisms behind stress-induced cellular responses. Full article
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26 pages, 4066 KiB  
Article
Identifying Endogenous Proteins of Perennial Ryegrass (Lolium perenne) with Ex Vivo Antioxidant Activity
by Kathrine Danner Aakjær Pedersen, Line Thopholm Andersen, Mads Heiselberg, Camilla Agerskov Brigsted, Freja Lyngs Støvring, Louise Mailund Mikkelsen, Sofie Albrekt Hansen, Christian Enrico Rusbjerg-Weberskov, Mette Lübeck and Simon Gregersen Echers
Proteomes 2025, 13(1), 8; https://doi.org/10.3390/proteomes13010008 - 5 Feb 2025
Viewed by 480
Abstract
Background: During the initial steps of green biorefining aimed at protein recovery, endogenous proteins and enzymes, along with, e.g., phytochemical constituents, are decompartmentalized into a green juice. This creates a highly dynamic environment prone to a plethora of reactions including oxidative protein [...] Read more.
Background: During the initial steps of green biorefining aimed at protein recovery, endogenous proteins and enzymes, along with, e.g., phytochemical constituents, are decompartmentalized into a green juice. This creates a highly dynamic environment prone to a plethora of reactions including oxidative protein modification and deterioration. Obtaining a fundamental understanding of the enzymes capable of exerting antioxidant activity ex vivo could help mitigate these reactions for improved product quality. Methods: In this study, we investigated perennial ryegrass (Lolium perenne var. Abosan 1), one of the most widely used turf and forage grasses, as a model system. Using size exclusion chromatography, we fractionated the green juice to investigate in vitro antioxidant properties and coupled this with quantitative bottom-up proteomics, GO-term analysis, and fraction-based enrichment. Results: Our findings revealed that several enzymes, such as superoxide dismutase and peroxiredoxin proteoforms, already known for their involvement in in vivo oxidative protection, are enriched in fractions displaying increased in vitro antioxidant activity, indicating retained activity ex vivo. Moreover, this study provides the most detailed characterization of the L. perenne proteome today and delivers new insights into protein-level partitioning during wet fractionation. Conclusions: Ultimately, this work contributes to a better understanding of the first steps of green biorefining and provides the basis for process optimization. Full article
(This article belongs to the Section Plant Proteomics)
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21 pages, 3652 KiB  
Article
Differential Signaling Pathways Identified in Aqueous Humor, Anterior Capsule, and Crystalline Lens of Age-Related, Diabetic, and Post-Vitrectomy Cataract
by Christina Karakosta, Martina Samiotaki, Anastasios Bisoukis, Konstantinos I. Bougioukas, George Panayotou, Dimitrios Papaconstantinou and Marilita M. Moschos
Proteomes 2025, 13(1), 7; https://doi.org/10.3390/proteomes13010007 - 3 Feb 2025
Viewed by 454
Abstract
Background: The purpose of this study was to detect proteomic alterations and corresponding signaling pathways involved in the formation of age-related cataract (ARC), diabetic cataract (DC), and post-vitrectomy cataract (PVC). Methods: Three sample types, the aqueous humor (AH), the anterior capsule [...] Read more.
Background: The purpose of this study was to detect proteomic alterations and corresponding signaling pathways involved in the formation of age-related cataract (ARC), diabetic cataract (DC), and post-vitrectomy cataract (PVC). Methods: Three sample types, the aqueous humor (AH), the anterior capsule (AC), and the content of the phaco cassette, were collected during phacoemulsification surgery. The samples were obtained from 12 participants without diabetes mellitus (DM), 11 participants with DM, and 7 participants without DM, with a history of vitrectomy surgery in the past 12 months. The Sp3 protocol (Single-Pot, Solid-Phase, Sample-Preparation) was used for the sample preparation. The recognition and quantification of proteins were carried out with liquid chromatography online with tandem mass spectrometry. The DIA-NN software was applied for the identification and quantification of peptides/proteins. Statistical analysis and data visualization were conducted on Perseus software. Data are available via ProteomeXchange. Results: A very rich atlas of the lens and AH proteome has been generated. Glycosaminoglycan biosynthesis and the non-canonical Wnt receptor signaling pathway were differentially expressed in ARC compared to both the DC and PVC groups. In the PVC group, complement activation was differentially expressed in AH samples, while glutathione metabolism and oxidoreductase activity were differentially expressed in AC samples. Microfilament motor activity, microtubule cytoskeleton organization, and microtubule binding were differentially expressed in the DC and PVC groups in both AH and AC samples. Conclusions: The results of this study expand the existing knowledge on pathways involved in the pathophysiology of cataract, and suggest possible important druggable targets for slower progression or even prevention of cataract. Full article
(This article belongs to the Special Issue Clinical Proteomics: Fourth Edition)
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20 pages, 3344 KiB  
Article
DNA Damage-Induced Ferroptosis: A Boolean Model Regulating p53 and Non-Coding RNAs in Drug Resistance
by Shantanu Gupta, Daner A. Silveira, José Carlos M. Mombach and Ronaldo F. Hashimoto
Proteomes 2025, 13(1), 6; https://doi.org/10.3390/proteomes13010006 - 20 Jan 2025
Viewed by 882
Abstract
The tumor suppressor p53, in its wild-type form, plays a central role in cellular homeostasis by regulating senescence, apoptosis, and autophagy within the DNA damage response (DDR). Recent findings suggest that wild-type p53 also governs ferroptosis, an iron-dependent cell death process driven by [...] Read more.
The tumor suppressor p53, in its wild-type form, plays a central role in cellular homeostasis by regulating senescence, apoptosis, and autophagy within the DNA damage response (DDR). Recent findings suggest that wild-type p53 also governs ferroptosis, an iron-dependent cell death process driven by lipid peroxidation. Post-translational modifications of p53 generate proteoforms that significantly enhance its functional diversity in regulating these mechanisms. A key target in this process is the cystine/glutamate transporter (xCT), which is essential for redox balance and ferroptosis resistance. Additionally, p53-induced miR-34c-5p suppresses cancer cell proliferation and drug resistance by modulating Myc, an oncogene further influenced by non-coding RNAs like circular RNA NOTCH1 (CricNOTCH1) and long non-coding RNA MALAT1. However, the exact role of these molecules in ferroptosis remains unclear. To address this, we introduce the first dynamic Boolean model that delineates the influence of these ncRNAs and p53 on ferroptosis, apoptosis, and senescence within the DDR context. Validated through gain- and loss-of-function perturbations, our model closely aligns with experimental observations in cancers such as oral squamous cell carcinoma, nasopharyngeal carcinoma, and osteosarcoma. The model identifies crucial positive feedback loops (CricNOTCH1/miR-34c/Myc, MALAT1/miR-34c/Myc, and Myc/xCT) and highlights the therapeutic potential of using p53 proteoforms and ncRNAs to combat drug resistance and induce cancer cell death. Full article
(This article belongs to the Section Multi-Omics Studies that Include Proteomics)
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5 pages, 175 KiB  
Editorial
Enhancing Biomedicine: Proteomics and Metabolomics in Action
by Michele Costanzo, Marianna Caterino and Lucia Santorelli
Proteomes 2025, 13(1), 5; https://doi.org/10.3390/proteomes13010005 - 16 Jan 2025
Viewed by 418
Abstract
The rapid and substantial advancements in proteomic and metabolomic technologies have revolutionized our ability to investigate biological systems [...] Full article
(This article belongs to the Topic Proteomics and Metabolomics in Biomedicine, 2nd Volume)
17 pages, 6539 KiB  
Article
Identification of Proteoforms Related to Nelumbo nucifera Flower Petaloid Through Proteogenomic Strategy
by Zhongyuan Lin, Jiantao Shu, Yu Qin, Dingding Cao, Jiao Deng and Pingfang Yang
Proteomes 2025, 13(1), 4; https://doi.org/10.3390/proteomes13010004 - 15 Jan 2025
Viewed by 652
Abstract
Nelumbo nucifera is an aquatic plant with a high ornamental value due to its flower. Despite the release of several versions of the lotus genome, its annotation remains inefficient, which makes it difficult to obtain a more comprehensive knowledge when –omic studies are [...] Read more.
Nelumbo nucifera is an aquatic plant with a high ornamental value due to its flower. Despite the release of several versions of the lotus genome, its annotation remains inefficient, which makes it difficult to obtain a more comprehensive knowledge when –omic studies are applied to understand the different biological processes. Focusing on the petaloid of the lotus flower, we conducted a comparative proteomic analysis among five major floral organs. The proteogenomic strategy was applied to analyze the mass spectrometry data in order to dig out novel proteoforms that are involved in the petaloids of the lotus flower. The results revealed that a total of 4863 proteins corresponding to novel genes were identified, with 227 containing single amino acid variants (SAAVs), and 72 originating from alternative splicing (AS) genes. In addition, a range of post-translational modifications (PTMs) events were also identified in lotus. Through functional annotation and homology analysis with 24 closely related plant species, we identified five candidate proteins associated with floral organ development, which were not identified by ordinary proteomic analysis. This study not only provides new insights into understanding the mechanism of petaloids in lotus but is also helpful in identifying new proteoforms to improve the annotation of the lotus genome. Full article
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26 pages, 3803 KiB  
Article
Novel Integration of Spatial and Single-Cell Omics Data Sets Enables Deeper Insights into IPF Pathogenesis
by Fei Wang, Liang Jin, Xue Wang, Baoliang Cui, Yingli Yang, Lori Duggan, Annette Schwartz Sterman, Sarah M. Lloyd, Lisa A. Hazelwood, Neha Chaudhary, Bhupinder Bawa, Lucy A. Phillips, Yupeng He and Yu Tian
Proteomes 2025, 13(1), 3; https://doi.org/10.3390/proteomes13010003 - 13 Jan 2025
Viewed by 957
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by repetitive alveolar injuries with excessive deposition of extracellular matrix (ECM) proteins. A crucial need in understanding IPF pathogenesis is identifying cell types associated with histopathological regions, particularly local fibrosis centers known as [...] Read more.
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by repetitive alveolar injuries with excessive deposition of extracellular matrix (ECM) proteins. A crucial need in understanding IPF pathogenesis is identifying cell types associated with histopathological regions, particularly local fibrosis centers known as fibroblast foci. To address this, we integrated published spatial transcriptomics and single-cell RNA sequencing (scRNA-seq) transcriptomics and adopted the Query method and the Overlap method to determine cell type enrichments in histopathological regions. Distinct fibroblast cell types are highly associated with fibroblast foci, and transitional alveolar type 2 and aberrant KRT5-/KRT17+ (KRT: keratin) epithelial cells are associated with morphologically normal alveoli in human IPF lungs. Furthermore, we employed laser capture microdissection-directed mass spectrometry to profile proteins. By comparing with another published similar dataset, common differentially expressed proteins and enriched pathways related to ECM structure organization and collagen processing were identified in fibroblast foci. Importantly, cell type enrichment results from innovative spatial proteomics and scRNA-seq data integration accord with those from spatial transcriptomics and scRNA-seq data integration, supporting the capability and versatility of the entire approach. In summary, we integrated spatial multi-omics with scRNA-seq data to identify disease-associated cell types and potential targets for novel therapies in IPF intervention. The approach can be further applied to other disease areas characterized by spatial heterogeneity. Full article
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21 pages, 2578 KiB  
Article
HRAMS Proteomics Insights on the Anti-Filarial Effect of Ocimum sanctum: Implications in Phytochemical-Based Drug-Targeting and Designing
by Ayushi Mishra, Vipin Kumar, Sunil Kumar, HariOm Singh and Anchal Singh
Proteomes 2025, 13(1), 2; https://doi.org/10.3390/proteomes13010002 - 27 Dec 2024
Viewed by 790
Abstract
Lymphatic filariasis (LF) continues to impact 657 million individuals worldwide, resulting in lifelong and chronic impairment. The prevalent anti-filarial medications—DEC, albendazole, and ivermectin—exhibit limited adulticidal efficacy. Despite ongoing LF eradication programs, novel therapeutic strategies are essential for effective control. This study examines the [...] Read more.
Lymphatic filariasis (LF) continues to impact 657 million individuals worldwide, resulting in lifelong and chronic impairment. The prevalent anti-filarial medications—DEC, albendazole, and ivermectin—exhibit limited adulticidal efficacy. Despite ongoing LF eradication programs, novel therapeutic strategies are essential for effective control. This study examines the mechanism of action of Ocimum sanctum on the filarial parasites Setaria cervi via a synergistic biochemical and proteomics methodology. The ethanolic extract of Ocimum sanctum (EOS) demonstrated potential anti-filarial action in the MTT reduction experiment, with an LC50 value of 197.24 µg/mL. After EOS treatment, an elevation in lipid peroxidation (51.92%), protein carbonylation (48.99%), and NADPH oxidase (88.88%) activity, along with a reduction in glutathione (GSH) (−39.23%), glutathione reductase (GR) (−60.17%), and glutathione S transferase (GST) (−50.48%) activity, was observed. The 2D gel electrophoresis identified 20 decreased and 11 increased protein spots in the EOS-treated parasites relative to the control group. Additionally, in drug docking analysis, the EOS bioactive substances ursolic acid, rutin, and rosmarinic acid show a significant binding affinity with the principal differentially expressed proteins. This paper demonstrates, for the first time, that the anti-filarial efficacy of EOS is primarily facilitated by its impact on energy metabolism, antioxidant mechanisms, and stress response systems of the parasites. Full article
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13 pages, 3918 KiB  
Article
Phospho-Proteomics Analysis of Early Response to X-Ray Irradiation Reveals Molecular Mechanism Potentially Related to U251 Cell Radioresistance
by Ousseynou Ben Diouf, Antoine Gilbert, Benoit Bernay, Randi G. Syljuåsen, Mihaela Tudor, Mihaela Temelie, Diana I. Savu, Mamadou Soumboundou, Cheikh Sall and François Chevalier
Proteomes 2025, 13(1), 1; https://doi.org/10.3390/proteomes13010001 - 25 Dec 2024
Viewed by 695
Abstract
Glioblastoma (GBM) is a devastating malignant brain tumor with a poor prognosis. GBM is associated with radioresistance. Post-translational modifications (PTMs) such as protein phosphorylation can play an important role in the cellular response to radiation. To better understand the early cellular activities after [...] Read more.
Glioblastoma (GBM) is a devastating malignant brain tumor with a poor prognosis. GBM is associated with radioresistance. Post-translational modifications (PTMs) such as protein phosphorylation can play an important role in the cellular response to radiation. To better understand the early cellular activities after radiation in GBM, we carried out a phospho-proteomic study on the U251 cell line 3 h after X-ray irradiation (6Gy) and on non-irradiated cells. Our study showed a strong modification of proteoform phosphorylation in response to radiation. We found 453 differentially expressed phosphopeptides (DEPs), with 211 being upregulated and 242 being downregulated. A GO enrichment analysis of DEPs showed a strong enrichment of the signaling pathways involved in DNA damage response after irradiation and categorized them into biological processes (BPs), cellular components (CCs) and molecular functions (MFs). Certain accessions such as BRCA1, MDC1, H2AX, MDC1, TP53BP1 were dynamically altered in our fraction and are highly associated with the signaling pathways enriched after radiation. Full article
(This article belongs to the Section Proteomics of Human Diseases and Their Treatments)
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