Proteomes

20 pages, 3344 KiB

Open AccessArticle

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

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

Open AccessEditorial

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

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

Open AccessArticle

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

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

Open AccessArticle

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

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

Open AccessArticle

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

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

Open AccessArticle

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

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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18 pages, 1870 KiB

Open AccessArticle

Affinity-Enriched Plasma Proteomics for Biomarker Discovery in Abdominal Aortic Aneurysms

by Nicolai Bjødstrup Palstrøm, Kristian Boje Nielsen, Amanda Jessica Campbell, Mette Soerensen, Lars Melholt Rasmussen, Jes Sanddal Lindholt and Hans Christian Beck

Proteomes 2024, 12(4), 37; https://doi.org/10.3390/proteomes12040037 - 9 Dec 2024

Abstract

Abdominal aortic aneurysm (AAA) is a life-threatening condition characterized by the weakening and dilation of the abdominal aorta. Few diagnostic biomarkers have been proposed for this condition. We performed mass spectrometry-based proteomics analysis of affinity-enriched plasma from 45 patients with AAA and 45 [...] Read more.

Abdominal aortic aneurysm (AAA) is a life-threatening condition characterized by the weakening and dilation of the abdominal aorta. Few diagnostic biomarkers have been proposed for this condition. We performed mass spectrometry-based proteomics analysis of affinity-enriched plasma from 45 patients with AAA and 45 matched controls to identify changes to the plasma proteome and potential diagnostic biomarkers. Gene ontology analysis revealed a significant upregulation of the proteins involved in inflammation, coagulation, and extracellular matrix in AAA patients, while proteins related to angiogenesis were among those downregulated. Using recursive feature elimination, we identified a subset of 10 significantly regulated proteins that were highly predictive of AAA. A random forest classifier trained on these proteins achieved an area under the curve (AUC) of 0.93 [95% CI: 0.91–0.95] using cross-validation. Further validation in a larger cohort is necessary to confirm these results. Full article

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11 pages, 1721 KiB

Open AccessArticle

Peptidomic Analysis Reveals Temperature-Dependent Proteolysis in Rainbow Trout (Oncorhynchus mykiss) Meat During Sous-Vide Cooking

by Miyu Sakuyama, Yuri Kominami and Hideki Ushio

Proteomes 2024, 12(4), 36; https://doi.org/10.3390/proteomes12040036 - 27 Nov 2024

Abstract

Sous vide, a cooking method that involves vacuum-sealed fish at low temperatures, yields a uniquely tender, easily flaked texture. Previous research on sous-vide tenderization has focused on thermal protein denaturation. On the other hand, the contribution of proteases, activated at low temperatures in [...] Read more.

Sous vide, a cooking method that involves vacuum-sealed fish at low temperatures, yields a uniquely tender, easily flaked texture. Previous research on sous-vide tenderization has focused on thermal protein denaturation. On the other hand, the contribution of proteases, activated at low temperatures in fish meat, has been suggested. However, the details of protein degradation remain unclear. This study employed SDS-PAGE/immunoblot and peptidomic analysis of rainbow trout to assess proteolysis during sous-vide cooking. The results from SDS-PAGE and immunoblot analysis indicated reduced thermal aggregation of sarcoplasmic proteins and increased depolymerization of actin under low-temperature cooking conditions. A comparison of the peptidome showed that the proteolysis of myofibrillar proteins was accelerated during sous-vide cooking, with distinct proteases potentially activated at different cooking temperatures. Terminome analysis revealed the contribution of specific proteases at higher temperatures in rainbow trout. The results of this study demonstrate the thermal denaturation of sarcoplasmic proteins and proteolysis of myofibrillar proteins in rainbow trout meat during sous-vide cooking and its temperature dependence. The methodology in the present study could provide insights into the optimization of cooking conditions for different fish species, potentially leading to improved texture and quality of sous-vide products. Full article

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10 pages, 1312 KiB

Open AccessTechnical Note

Deep Proteome Coverage of Microglia Using a Streamlined Data-Independent Acquisition-Based Proteomic Workflow: Method Consideration for a Phenotypically Diverse Cell Type

by Jessica Wohlfahrt, Jennifer Guergues and Stanley M. Stevens, Jr.

Proteomes 2024, 12(4), 35; https://doi.org/10.3390/proteomes12040035 - 27 Nov 2024

Abstract

As the primary innate immune cells of the brain, microglia play a key role in various homeostatic and disease-related processes. To carry out their numerous functions, microglia adopt a wide range of phenotypic states. The proteomic landscape represents a more accurate molecular representation [...] Read more.

As the primary innate immune cells of the brain, microglia play a key role in various homeostatic and disease-related processes. To carry out their numerous functions, microglia adopt a wide range of phenotypic states. The proteomic landscape represents a more accurate molecular representation of these phenotypes; however, microglia present unique challenges for proteomic analysis. This study implemented a streamlined liquid- and gas-phase fractionation method with data-dependent acquisition (DDA) and parallel accumulation–serial fragmentation (PASEF) analysis on a TIMS-TOF instrument to compile a comprehensive protein library obtained from adult-derived, immortalized mouse microglia with low starting material (10 µg). The empirical library consisted of 9140 microglial proteins and was utilized to identify an average of 7264 proteins/run from single-shot, data-independent acquisition (DIA)-based analysis microglial cell lysate digest (200 ng). Additionally, a predicted library facilitated the identification of 7519 average proteins/run from the same DIA data, revealing complementary coverage compared with the empirical library and collectively increasing coverage to approximately 8000 proteins. Importantly, several microglia-relevant pathways were uniquely identified with the empirical library approach. Overall, we report a simplified, reproducible approach to address the proteome complexity of microglia using low sample input and show the importance of library optimization for this phenotypically diverse cell type. Full article

(This article belongs to the Section Proteomics Technology and Methodology Development)

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12 pages, 799 KiB

Open AccessArticle

The Non-Linear Profile of Aging: U-Shaped Expression of Myostatin, Follistatin and Intermediate Signals in a Longitudinal In Vitro Murine Cell Sarcopenia Model

by Janire Alonso-Puyo, Oihane Izagirre-Fernandez, Olatz Crende, Jesús Seco-Calvo, Ainhoa Fernandez-Atutxa, Diego Fernandez-Lazaro, Patricia Garcia-Gallastegi and Begoña Sanz

Proteomes 2024, 12(4), 34; https://doi.org/10.3390/proteomes12040034 - 22 Nov 2024

Abstract

Sarcopenia is linked to the decline in muscle mass, strength and function during aging. It affects the quality and life expectancy and can lead to dependence. The biological process underlying sarcopenia is unclear, but the proteins myostatin and follistatin are involved in the [...] Read more.

Sarcopenia is linked to the decline in muscle mass, strength and function during aging. It affects the quality and life expectancy and can lead to dependence. The biological process underlying sarcopenia is unclear, but the proteins myostatin and follistatin are involved in the balance between muscle breakdown and synthesis. While myostatin promotes muscle breakdown, follistatin promotes muscle growth, but several works have shown an inconsistent association of these proteins with aging-related parameters in serum of older people. We aimed to know the evolution of these putative sarcopenia biomarkers along muscle aging in an in vitro model. We created and phenotyped a longitudinal murine model (C2C12 cells). Then, we analyzed the protein and genetic expression of myostatin and follistatin as well as the signaling pathway regulators mTOR and RPS6KB1. Myostatin and RPS6KB1 showed a similar tendency in both protein and genetic expression with aging (basal–up–down). Follistatin, on the other hand, shows the opposite tendency (basal–down–up). Regarding mTOR, the tendencies differ when analyzing proteins (basal–up–down) or genes (basal–down–down). Our work demonstrates a U-shape tendency for myostatin and follistatin and for the signaling pathway regulators. These results could be of the utmost importance when designing further research on seeking molecular biomarkers and/or targets for sarcopenia. Full article

(This article belongs to the Section Identification of Potential Biomarkers and Potential Therapeutic Targets)

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17 pages, 3899 KiB

Open AccessArticle

Assessment of Data-Independent Acquisition Mass Spectrometry (DIA-MS) for the Identification of Single Amino Acid Variants

by Ivo Fierro-Monti, Klemens Fröhlich, Christian Schori and Alexander Schmidt

Proteomes 2024, 12(4), 33; https://doi.org/10.3390/proteomes12040033 - 6 Nov 2024

Abstract

Proteogenomics integrates genomic and proteomic data to elucidate cellular processes by identifying variant peptides, including single amino acid variants (SAAVs). In this study, we assessed the capability of data-independent acquisition mass spectrometry (DIA-MS) to identify SAAV peptides in HeLa cells using various search [...] Read more.

Proteogenomics integrates genomic and proteomic data to elucidate cellular processes by identifying variant peptides, including single amino acid variants (SAAVs). In this study, we assessed the capability of data-independent acquisition mass spectrometry (DIA-MS) to identify SAAV peptides in HeLa cells using various search engine pipelines. We developed a customised sequence database (DB) incorporating SAAV sequences from the HeLa genome and conducted searches using DIA-NN, Spectronaut, and Fragpipe-MSFragger. Our evaluation focused on identifying true positive SAAV peptides and false positives through entrapment DBs. This study revealed that DIA-MS provides reproducible and comprehensive coverage of the proteome, identifying a substantial proportion of SAAV peptides. Notably, the DIA-MS searches maintained consistent identification of SAAV peptides despite varying sizes of the entrapment DB. A comparative analysis showed that Fragpipe-MSFragger (FP-DIA) demonstrated the most conservative and effective performance, exhibiting the lowest false discovery match ratio (FDMR). Additionally, integrating DIA and data-dependent acquisition (DDA) MS data search outputs enhanced SAAV peptide identification, with a lower false discovery rate (FDR) observed in DDA searches. The validation using stable isotope dilution and parallel reaction monitoring (SID-PRM) confirmed the SAAV peptides identified by DIA-MS and DDA-MS searches, highlighting the reliability of our approach. Our findings underscore the effectiveness of DIA-MS in proteogenomic workflows for identifying SAAV peptides, offering insights into optimising search engine pipelines and DB construction for accurate proteomics analysis. These methodologies advance the understanding of proteome variability, contributing to cancer research and the identification of novel proteoform therapeutic targets. Full article

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19 pages, 13454 KiB

Open AccessArticle

Transcriptomics Revealed Differentially Expressed Transcription Factors and MicroRNAs in Human Diabetic Foot Ulcers

by Vikrant Rai

Proteomes 2024, 12(4), 32; https://doi.org/10.3390/proteomes12040032 - 5 Nov 2024

Abstract

Non-healing diabetic foot ulcers (DFUs) not only significantly increase morbidity and mortality but also cost a lot and drain healthcare resources. Persistent inflammation, decreased angiogenesis, and altered extracellular matrix remodeling contribute to delayed healing or non-healing. Recent studies suggest an increasing trend of [...] Read more.

Non-healing diabetic foot ulcers (DFUs) not only significantly increase morbidity and mortality but also cost a lot and drain healthcare resources. Persistent inflammation, decreased angiogenesis, and altered extracellular matrix remodeling contribute to delayed healing or non-healing. Recent studies suggest an increasing trend of DFUs in diabetes patients, and non-healing DFYs increase the incidence of amputation. Despite the current treatment with offloading, dressing, antibiotics use, and oxygen therapy, the risk of amputation persists. Thus, there is a need to understand the molecular and cellular factors regulating healing in DFUs. The ongoing research based on proteomics and transcriptomics has predicted multiple potential targets, but there is no definitive therapy to enhance healing in chronic DFUs. Increased or decreased expression of various proteins encoded by genes, whose expression transcriptionally and post-transcriptionally is regulated by transcription factors (TFs) and microRNAs (miRs), regulates DFU healing. For this study, RNA sequencing was conducted on 20 DFU samples of ulcer tissue and non-ulcerated nearby healthy tissues. The IPA analysis revealed various activated and inhibited transcription factors and microRNAs. Further network analysis revealed interactions between the TFs and miRs and the molecular targets of these TFs and miRs. The analysis revealed 30 differentially expressed transcription factors (21 activated and 9 inhibited), two translational regulators (RPSA and EIF4G2), and seven miRs, including mir-486, mir-324, mir-23, mir-186, mir-210, mir-199, and mir-338 in upstream regulators (p < 0.05), while causal network analysis (p < 0.05) revealed 28 differentially expressed TFs (19 activated and 9 inhibited), two translational regulators (RPSA and EIF4G2), and five miRs including mir-155, mir-486, mir-324, mir-210, and mir-1225. The protein–protein interaction analysis revealed the interaction of various novel proteins with the proteins involved in regulating DFU pathogenesis and healing. The results of this study highlight many activated and inhibited novel TFs and miRs not reported in the literature so far, as well as the targeted molecules. Since proteins are the functional units during biological processes, alteration of gene expression may result in different proteoforms and protein species, making the wound microenvironment a complex protein interaction (proteome complexity). Thus, investigating the effects of these TFs and miRs on protein expression using proteomics and combining these results with transcriptomics will help advance research on DFU healing and delineate potential therapeutic strategies. Full article

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13 pages, 1617 KiB

Open AccessArticle

Comparative Proteome-Wide Abundance Profiling of Yeast Strains Deleted for Cdc48 Adaptors

by Valentina Rossio and Joao A. Paulo

Proteomes 2024, 12(4), 31; https://doi.org/10.3390/proteomes12040031 - 30 Oct 2024

Abstract

The yeast ATPase Cdc48 (known as p97/VCP in human cells) plays an important role in the Ubiquitin Proteasome System. VCP is essential for cancer cell proliferation, and its dysregulation has been implicated in several neurodegenerative diseases. Cdc48 functions by extracting ubiquitylated proteins from [...] Read more.

The yeast ATPase Cdc48 (known as p97/VCP in human cells) plays an important role in the Ubiquitin Proteasome System. VCP is essential for cancer cell proliferation, and its dysregulation has been implicated in several neurodegenerative diseases. Cdc48 functions by extracting ubiquitylated proteins from membranes, protein complexes and chromatin by often facilitating their proteasomal degradation. Specific adaptors or cofactors, primarily belonging to the UBX domain-containing protein family (which has seven members in Saccharomyces cerevisiae) recruit Cdc48 to ubiquitylated proteins. Here, we employed sample multiplexing-based quantitative mass spectrometry to profile global protein abundance in p97 adaptor deletion strains, specifically comparing seven single deletion strains of UBX domain-containing proteins and the Cuz1 deletion strain, which belongs to the zinc finger AN1-type domain protein family. We observed that each strain showed unique sets of differentially abundant proteins compared to the wild type. Our analysis also revealed a role for Ubx3 in maintaining wild type levels of mitochondrial proteins. Overall, we identified ~1400 differentially abundant proteins in the absence of a specific Cdc48 adaptor. This unique dataset offers a valuable resource for studying the functions of these adaptors, aiming to achieve a better understanding of the cellular processes regulated by Cdc48 itself and to deepen our understanding of the Ubiquitin Proteasome System. Full article

(This article belongs to the Section Microbial Proteomics)

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17 pages, 2555 KiB

Open AccessArticle

Multiple Reaction Monitoring–Mass Spectrometric Immunoassay Analysis of Parathyroid Hormone Fragments with Vitamin D Deficiency in Patients with Diabetes Mellitus

by Hicham Benabdelkamel, Refat M. Nimer, Afshan Masood, Maha Al Mogren, Anas M. Abdel Rahman and Assim A. Alfadda

Proteomes 2024, 12(4), 30; https://doi.org/10.3390/proteomes12040030 - 14 Oct 2024

Abstract

Current immunoassay techniques for analyzing clinically relevant parathyroid hormone (PTH) circulating fragments cannot distinguish microheterogeneity among structurally similar molecular species. This hinders the identification of molecular species and the capture of target analyte information. Since structural modifications are important in disease pathways, mass [...] Read more.

Current immunoassay techniques for analyzing clinically relevant parathyroid hormone (PTH) circulating fragments cannot distinguish microheterogeneity among structurally similar molecular species. This hinders the identification of molecular species and the capture of target analyte information. Since structural modifications are important in disease pathways, mass spectrometry can detect, identify, and quantify heterogeneous ligands captured by antibodies. We aimed to create a sensitive and selective multiple reaction monitoring–mass spectrometric immunoassay analysis (MRM-MSIA)-based method for detecting and quantifying PTH fragments or proteoforms for clinical research. Our study established MRM transitions using triple-quadrupole tandem mass spectrometry for the signature peptides of five PTH fragments. This method was validated according to FDA guidelines, employing the mass spectrometric immunoassay (MSIA) protocol to bolster detection selectivity and sensitivity. This validated approach was applied by analyzing samples from type 2 diabetes mellitus (T2DM) patients with and without vitamin D deficiency. We found serum PTH fragments associated with vitamin D deficiency in patients with and without T2DM. We developed and validated the MRM-MSIA technique specifically designed for the detection and quantification (amino acid (aa38–44), (aa45–51), and (aa65–75)) of these fragments associated with vitamin D deficiency and T2DM. This study is the first to accurately quantify plasma PTH fragments using MRM-MSIA, demonstrating its potential for clinical diagnostics. Full article

(This article belongs to the Section Proteomics Technology and Methodology Development)

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12 pages, 4636 KiB

Open AccessArticle

Circulating Factors as Potential Biomarkers of Cardiovascular Damage Progression Associated with Type 2 Diabetes

by Giovanni Sartore, Francesco Piarulli, Eugenio Ragazzi, Alice Mallia, Stefania Ghilardi, Massimo Carollo, Annunziata Lapolla and Cristina Banfi

Proteomes 2024, 12(4), 29; https://doi.org/10.3390/proteomes12040029 - 11 Oct 2024

Cited by 1

Abstract

Background: Diabetes, particularly type 2 diabetes (T2D), is linked with an increased risk of developing coronary heart disease (CHD). The present study aimed to evaluate potential circulating biomarkers of CHD by adopting a targeted proteomic approach based on proximity extension assays (PEA). [...] Read more.

Background: Diabetes, particularly type 2 diabetes (T2D), is linked with an increased risk of developing coronary heart disease (CHD). The present study aimed to evaluate potential circulating biomarkers of CHD by adopting a targeted proteomic approach based on proximity extension assays (PEA). Methods: The study was based on 30 patients with both T2D and CHD (group DC), 30 patients with T2D without CHD (group DN) and 29 patients without diabetes but with a diagnosis of CHD (group NC). Plasma samples were analyzed using PEA, with an Olink Target 96 cardiometabolic panel expressed as normalized protein expression (NPX) units. Results: Lysosomal Pro-X carboxypeptidase (PRCP), Liver carboxylesterase 1 (CES1), Complement C2 (C2), and Intercellular adhesion molecule 3 (ICAM3) were lower in the DC and NC groups compared with the DN groups. Lithostathine-1-alpha (REG1A) and Immunoglobulin lambda constant 2 (IGLC2) were found higher in the DC group compared to DN and NC groups. ROC analysis suggested a significant ability of the six proteins to distinguish among the three groups (whole model test p < 0.0001, AUC 0.83–0.88), with a satisfactory discriminating performance in terms of sensitivity (77–90%) and specificity (70–90%). A possible role of IGLC2, PRCP, and REG1A in indicating kidney impairment was found, with a sensitivity of 92% and specificity of 83%. Conclusions: The identified panel of six plasma proteins, using a targeted proteomic approach, provided evidence that these parameters could be considered in the chronic evolution of T2D and its complications. Full article

(This article belongs to the Topic Proteomics and Metabolomics in Biomedicine, 2nd Volume)

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16 pages, 10941 KiB

Open AccessArticle

Towards Characterization of Hass Avocado Peel and Pulp Proteome during Postharvest Shelf Life

by Carolina Camacho-Vázquez, José Miguel Elizalde-Contreras, Francisco Antonio Reyes-Soria, Juan Luis Monribot-Villanueva, José Antonio Guerrero-Analco, Janet Juarez-Escobar, Olinda Velázquez-López, Thuluz Meza-Menchaca, Esaú Bojórquez-Velázquez, Jesús Alejandro Zamora-Briseño, Monica Ramirez-Vazquez, Guadalupe Alheli González Barrenechea, Enrique Ibarra-Laclette and Eliel Ruiz-May

Proteomes 2024, 12(4), 28; https://doi.org/10.3390/proteomes12040028 - 28 Sep 2024

Abstract

In recent years, avocados have gained worldwide popularity as a nutritive food. This trend is causing a rise in the production of this fruit, which is accompanied by several problems associated with monocultural practices. Despite massive economic gains, limited molecular and structural information [...] Read more.

In recent years, avocados have gained worldwide popularity as a nutritive food. This trend is causing a rise in the production of this fruit, which is accompanied by several problems associated with monocultural practices. Despite massive economic gains, limited molecular and structural information has been generated about avocado ripening. In fact, limited studies have attempted to unravel the proteome complexity dynamics of avocado fruit. We therefore conducted a comparative proteomics study on avocado peel and pulp during the postharvest shelf life using tandem mass tag synchronous precursor selection triple-stage mass spectrometry. We identified 3161 and 1128 proteins in the peel and pulp, respectively. Peels exhibited major over-accumulation of proteins associated with water deprivation and oxidative stress, along with abscisic acid biosynthesis. Ethylene, jasmonic acid, phenylpropanoid, and flavonoid biosynthesis pathways were activated. Structurally, we observed the accumulation of lignin and a reduction in cuticular thickness, which coincides with the reduction in the levels of long-chain acyl-coenzyme A synthetase and a marginal increase in 10,16-dihydroxyhexadecanoic acid. Our study sheds light on the association of proteome modulation with the structural features of Hass avocado. Its detailed characterization will provide an alternative for better preservation during the postharvest period. Full article

(This article belongs to the Section Plant Proteomics)

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14 pages, 7429 KiB

Open AccessArticle

Protein Extraction Methods Suitable for Muscle Tissue Proteomic Analysis

by Lorenza Vantaggiato, Claudia Landi, Enxhi Shaba, Daniela Rossi, Vincenzo Sorrentino and Luca Bini

Proteomes 2024, 12(4), 27; https://doi.org/10.3390/proteomes12040027 - 25 Sep 2024

Abstract

Muscle tissue is one of the most dynamic and plastic tissues of the mammalian body and covers different roles, such as force generation and metabolic control. Muscular proteomics provides an important opportunity to reveal the molecular mechanisms behind muscle pathophysiology. To ensure successful [...] Read more.

Muscle tissue is one of the most dynamic and plastic tissues of the mammalian body and covers different roles, such as force generation and metabolic control. Muscular proteomics provides an important opportunity to reveal the molecular mechanisms behind muscle pathophysiology. To ensure successful proteomic analysis, it is necessary to have an efficient and reproducible protein extraction method. This study aimed to evaluate the efficacy of two different extraction protocols of muscle samples for two-dimensional gel electrophoresis. In particular, mouse muscle proteins were extracted by an SDS-based buffer (Method A) and by a UREA/CHAPS/DTE/TRIS solution (Method B). The efficacies of the methods were assessed by performing an image analysis of the 2DE gels and by statistical and multivariate analyses. The 2DE gels in both preparations showed good resolution and good spot overlapping. Methods A and B produced 2DE gels with different means of total spots, higher for B. Image analysis showed different patterns of protein abundance between the protocols. The results showed that the two methods extract and solubilize proteins with different chemical–physical characteristics and different cellular localizations. These results attest the efficacy and reproducibility of both protein extraction methods, which can be parallelly applied for comprehensive proteomic profiling of muscle tissue. Full article

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15 pages, 862 KiB

Open AccessReview

Multi-Omic Approaches in Cancer-Related Micropeptide Identification

by Katarina Vrbnjak and Raj Nayan Sewduth

Proteomes 2024, 12(3), 26; https://doi.org/10.3390/proteomes12030026 - 13 Sep 2024

Abstract

Despite the advances in modern cancer therapy, malignant diseases are still a leading cause of morbidity and mortality worldwide. Conventional treatment methods frequently lead to side effects and drug resistance in patients, highlighting the need for novel therapeutic approaches. Recent findings have identified [...] Read more.

Despite the advances in modern cancer therapy, malignant diseases are still a leading cause of morbidity and mortality worldwide. Conventional treatment methods frequently lead to side effects and drug resistance in patients, highlighting the need for novel therapeutic approaches. Recent findings have identified the existence of non-canonical micropeptides, an additional layer of the proteome complexity, also called the microproteome. These small peptides are a promising class of therapeutic agents with the potential to address the limitations of current cancer treatments. The microproteome is encoded by regions of the genome historically annotated as non-coding, and its existence has been revealed thanks to recent advances in proteomic and bioinformatic technology, which dramatically improved the understanding of proteome complexity. Micropeptides have been shown to be biologically active in several cancer types, indicating their therapeutic role. Furthermore, they are characterized by low toxicity and high target specificity, demonstrating their potential for the development of better tolerated drugs. In this review, we survey the current landscape of known micropeptides with a role in cancer progression or treatment, discuss their potential as anticancer agents, and describe the methodological challenges facing the proteome field of research. Full article

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30 pages, 1134 KiB

Open AccessReview

Transforming Clinical Research: The Power of High-Throughput Omics Integration

by Rui Vitorino

Proteomes 2024, 12(3), 25; https://doi.org/10.3390/proteomes12030025 - 6 Sep 2024

Abstract

High-throughput omics technologies have dramatically changed biological research, providing unprecedented insights into the complexity of living systems. This review presents a comprehensive examination of the current landscape of high-throughput omics pipelines, covering key technologies, data integration techniques and their diverse applications. It looks [...] Read more.

High-throughput omics technologies have dramatically changed biological research, providing unprecedented insights into the complexity of living systems. This review presents a comprehensive examination of the current landscape of high-throughput omics pipelines, covering key technologies, data integration techniques and their diverse applications. It looks at advances in next-generation sequencing, mass spectrometry and microarray platforms and highlights their contribution to data volume and precision. In addition, this review looks at the critical role of bioinformatics tools and statistical methods in managing the large datasets generated by these technologies. By integrating multi-omics data, researchers can gain a holistic understanding of biological systems, leading to the identification of new biomarkers and therapeutic targets, particularly in complex diseases such as cancer. The review also looks at the integration of omics data into electronic health records (EHRs) and the potential for cloud computing and big data analytics to improve data storage, analysis and sharing. Despite significant advances, there are still challenges such as data complexity, technical limitations and ethical issues. Future directions include the development of more sophisticated computational tools and the application of advanced machine learning techniques, which are critical for addressing the complexity and heterogeneity of omics datasets. This review aims to serve as a valuable resource for researchers and practitioners, highlighting the transformative potential of high-throughput omics technologies in advancing personalized medicine and improving clinical outcomes. Full article

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