Proteomes

9 pages, 1301 KiB

Open AccessArticle

Viral Biomarker Detection and Validation Using MALDI Mass Spectrometry Imaging (MSI)

by Matthew B. O’Rourke, Ben R. Roediger, Christopher J. Jolly, Ben Crossett, Matthew P. Padula and Phillip M. Hansbro

Proteomes 2022, 10(3), 33; https://doi.org/10.3390/proteomes10030033 - 13 Sep 2022

Cited by 1 | Viewed by 2343

Abstract

(1) Background: MALDI imaging is a technique that still largely depends on time of flight (TOF)-based instrument such as the Bruker UltrafleXtreme. While capable of performing targeted MS/MS, these instruments are unable to perform fragmentation while imaging a tissue section necessitating the reliance [...] Read more.

(1) Background: MALDI imaging is a technique that still largely depends on time of flight (TOF)-based instrument such as the Bruker UltrafleXtreme. While capable of performing targeted MS/MS, these instruments are unable to perform fragmentation while imaging a tissue section necessitating the reliance of MS1 values for peptide level identifications. With this premise in mind, we have developed a hybrid bioinformatic/image-based method for the identification and validation of viral biomarkers. (2) Methods: Formalin-Fixed Paraffin-Embedded (FFPE) mouse samples were sectioned, mounted and prepared for mass spectrometry imaging using our well-established methods. Peptide identification was achieved by first extracting confident images corresponding to theoretical viral peptides. Next, those masses were used to perform a Peptide Mmass Fingerprint (PMF) searched against known viral FASTA sequences against a background mouse FASTA database. Finally, a correlational analysis was performed with imaging data to confirm pixel-by-pixel colocalization and intensity of viral peptides. (3) Results: 14 viral peptides were successfully identified with significant PMF Scores and a correlational result of >0.79 confirming the presence of the virus and distinguishing it from the background mouse proteins. (4) Conclusions: this novel approach leverages the power of mass spectrometry imaging and provides confident identifications for viral proteins without requiring MS/MS using simple MALDI Time Of Flight/Time Of Flight (TOF/TOF) instrumentation. Full article

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17 pages, 1229 KiB

Open AccessReview

Proteomic Applications in Aquatic Environment Studies

by Nadeeka Thushari Gajahin Gamage, Rina Miyashita, Kazutaka Takahashi, Shuichi Asakawa and Jayan Duminda Mahesh Senevirathna

Proteomes 2022, 10(3), 32; https://doi.org/10.3390/proteomes10030032 - 01 Sep 2022

Cited by 5 | Viewed by 2854

Abstract

Genome determines the unique individualities of organisms; however, proteins play significant roles in the generation of the colorful life forms below water. Aquatic systems are usually complex and multifaceted and can take on unique modifications and adaptations to environmental changes by altering proteins [...] Read more.

Genome determines the unique individualities of organisms; however, proteins play significant roles in the generation of the colorful life forms below water. Aquatic systems are usually complex and multifaceted and can take on unique modifications and adaptations to environmental changes by altering proteins at the cellular level. Proteomics is an essential strategy for exploring aquatic ecosystems due to the diverse involvement of proteins, proteoforms, and their complexity in basic and advanced cellular functions. Proteomics can expedite the analysis of molecular mechanisms underlying biological processes in an aquatic environment. Previous proteomic studies on aquatic environments have mainly focused on pollution assessments, ecotoxicology, their role in the food industry, and extraction and identification of natural products. Aquatic protein biomarkers have been comprehensively reported and are currently extensively applied in the pharmaceutical and medical industries. Cellular- and molecular-level responses of organisms can be used as indicators of environmental changes and stresses. Conversely, environmental changes are expedient in predicting aquatic health and productivity, which are crucial for ecosystem management and conservation. Recent advances in proteomics have contributed to the development of sustainable aquaculture, seafood safety, and high aquatic food production. Proteomic approaches have expanded to other aspects of the aquatic environment, such as protein fingerprinting for species identification. In this review, we encapsulated current proteomic applications and evaluated the potential strengths, weaknesses, opportunities, and threats of proteomics for future aquatic environmental studies. The review identifies both pros and cons of aquatic proteomics and projects potential challenges and recommendations. We postulate that proteomics is an emerging, powerful, and integrated omics approach for aquatic environmental studies. Full article

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17 pages, 3802 KiB

Open AccessArticle

Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking Proteomics

by A. D. A. Shahinuzzaman, Abu Hena Mostafa Kamal, Jayanta K. Chakrabarty, Aurchie Rahman and Saiful M. Chowdhury

Proteomes 2022, 10(3), 31; https://doi.org/10.3390/proteomes10030031 - 01 Sep 2022

Cited by 1 | Viewed by 2664

Abstract

Toll-like receptor 4 (TLR4) is a receptor on an immune cell that can recognize the invasion of bacteria through their attachment with bacterial lipopolysaccharides (LPS). Hence, LPS is a pro-immune response stimulus. On the other hand, statins are lipid-lowering drugs and can also [...] Read more.

Toll-like receptor 4 (TLR4) is a receptor on an immune cell that can recognize the invasion of bacteria through their attachment with bacterial lipopolysaccharides (LPS). Hence, LPS is a pro-immune response stimulus. On the other hand, statins are lipid-lowering drugs and can also lower immune cell responses. We used human embryonic kidney (HEK 293) cells engineered to express HA-tagged TLR-4 upon treatment with LPS, statin, and both statin and LPS to understand the effect of pro- and anti-inflammatory responses. We performed a monoclonal antibody (mAb) directed co-immunoprecipitation (CO-IP) of HA-tagged TLR4 and its interacting proteins in the HEK 293 extracted proteins. We utilized an ETD cleavable chemical cross-linker to capture weak and transient interactions with TLR4 protein. We tryptic digested immunoprecipitated and cross-linked proteins on beads, followed by liquid chromatography–mass spectrometry (LC-MS/MS) analysis of the peptides. Thus, we utilized the label-free quantitation technique to measure the relative expression of proteins between treated and untreated samples. We identified 712 proteins across treated and untreated samples and performed protein network analysis using Ingenuity Pathway Analysis (IPA) software to reveal their protein networks. After filtering and evaluating protein expression, we identified macrophage myristoylated alanine-rich C kinase substrate (MARCKSL1) and creatine kinase proteins as a potential part of the inflammatory networks of TLR4. The results assumed that MARCKSL1 and creatine kinase proteins might be associated with a statin-induced anti-inflammatory response due to possible interaction with the TLR4. Full article

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15 pages, 2481 KiB

Open AccessArticle

Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure

by Fábio Trindade, Ana F. Ferreira, Francisca Saraiva, Diana Martins, Vera M. Mendes, Carla Sousa, Cristina Gavina, Adelino Leite-Moreira, Bruno Manadas, Inês Falcão-Pires and Rui Vitorino

Proteomes 2022, 10(3), 30; https://doi.org/10.3390/proteomes10030030 - 01 Sep 2022

Viewed by 4380

Abstract

The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenization and protein extraction are made difficult by tissue calcification. The reproducibility of proteome [...] Read more.

The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenization and protein extraction are made difficult by tissue calcification. The reproducibility of proteome studies is key in clinical translation of the findings. Thus, we aimed to optimize a protocol for aortic valve homogenization and protein extraction and to develop a standard operating procedure (SOP), which researchers can use to maximize protein yield while reducing inter-laboratory variability. We have compared the protein yield between conventional tissue grinding in nitrogen followed by homogenization with a Potter apparatus with a more advanced bead-beating system. Once we confirmed the superiority of the latter, we further optimized it by testing the effect of beads size, the number of homogenization cycles, tube capacity, lysis buffer/tissue mass ratio, and two different lysis buffers. Optimal protein extraction was achieved with 2.8 mm zirconium dioxide beads, in two homogenization cycles, in the presence of 20 µL RIPA buffer/mg tissue, using 2 mL O-ring cryotubes. As a proof of concept of the usefulness of this SOP for proteomics, the AV proteome of men and women with aortic stenosis was characterized, resulting in the quantification of proteins across six orders of magnitude and uncovering some putative proteins dysregulated by sex. Full article

(This article belongs to the Special Issue Proteomics of Body Fluids: Principles, Methods, and Applications)

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18 pages, 1392 KiB

Open AccessReview

Potential Satellite Cell-Linked Biomarkers in Aging Skeletal Muscle Tissue: Proteomics and Proteogenomics to Monitor Sarcopenia

by Diego Fernández-Lázaro, Evelina Garrosa, Jesús Seco-Calvo and Manuel Garrosa

Proteomes 2022, 10(3), 29; https://doi.org/10.3390/proteomes10030029 - 19 Aug 2022

Cited by 20 | Viewed by 3929

Abstract

Sarcopenia (Sp) is the loss of skeletal muscle mass associated with aging which causes an involution of muscle function and strength. Satellite cells (Sc) are myogenic stem cells, which are activated by injury or stress, and repair muscle tissue. With advancing age, there [...] Read more.

Sarcopenia (Sp) is the loss of skeletal muscle mass associated with aging which causes an involution of muscle function and strength. Satellite cells (Sc) are myogenic stem cells, which are activated by injury or stress, and repair muscle tissue. With advancing age, there is a decrease in the efficiency of the regenerative response of Sc. Diagnosis occurs with the Sp established by direct assessments of muscle. However, the detection of biomarkers in real-time biofluids by liquid biopsy could represent a step-change in the understanding of the molecular biology and heterogeneity of Sp. A total of 13 potential proteogenomic biomarkers of Sp by their physiological and biological interaction with Sc have been previously described in the literature. Increases in the expression of GDF11, PGC-1α, Sirt1, Pax7, Pax3, Myf5, MyoD, CD34, MyoG, and activation of Notch signaling stimulate Sc activity and proliferation, which could modulate and delay Sp progression. On the contrary, intensified expression of GDF8, p16INK4a, Mrf4, and activation of the Wnt pathway would contribute to early Sp development by directly inducing reduced and/or altered Sc function, which would attenuate the restorative capacity of skeletal muscle. Additionally, tissue biopsy remains an important diagnostic tool. Proteomic profiling of aged muscle tissues has shown shifts toward protein isoforms characteristic of a fast-to-slow transition process and an elevated number of oxidized proteins. In addition, a strong association between age and plasma values of growth differentiation factor 15 (GDF-15) has been described and serpin family A member 3 (serpin A3n) was more secreted by atrophied muscle cells. The identification of these new biomarkers holds the potential to change personalized medicine because it could predict in real time the course of Sp by monitoring its evolution and assessing responses to potential therapeutic strategies. Full article

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

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15 pages, 3337 KiB

Open AccessArticle

Disturbance of Key Cellular Subproteomes upon Propofol Treatment Is Associated with Increased Permeability of the Blood-Brain Barrier

by Timo Längrich, Kaya Bork, Rüdiger Horstkorte, Veronika Weber, Britt Hofmann, Matt Fuszard and Heidi Olzscha

Proteomes 2022, 10(3), 28; https://doi.org/10.3390/proteomes10030028 - 15 Aug 2022

Cited by 1 | Viewed by 2307

Abstract

Background: Propofol is a short-acting anesthetic, which is often used for induction and maintenance of general anesthesia, sedation for mechanically ventilated adults and procedural sedation. Several side effects of propofol are known and a substantial number of patients suffer from post-operative delirium after [...] Read more.

Background: Propofol is a short-acting anesthetic, which is often used for induction and maintenance of general anesthesia, sedation for mechanically ventilated adults and procedural sedation. Several side effects of propofol are known and a substantial number of patients suffer from post-operative delirium after propofol application. In this study, we analyzed the effect of propofol on the function and protein expression profile on a proteome-wide scale. Methods: We cultured human brain microvascular endothelial cells in absence and presence of propofol and analyzed the permeability of the blood-brain barrier (BBB) by fluorescein passage and protein abundance on a proteome-wide scale by mass spectrometry. Results: Propofol interfered with the function of the blood-brain barrier. This was not due to decreased adhesion of propofol-treated human brain microvascular endothelial cells. The proteomic analysis revealed that some key pathways in these cells were disturbed, such as oxygen metabolism, DNA damage recognition and response to stress. Conclusions: Propofol has strong effects on protein expression which could explain several side effects of propofol. Full article

(This article belongs to the Special Issue Blood-Brain Barrier Proteomics)

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14 pages, 1722 KiB

Open AccessReview

Metabolomics in Team-Sport Athletes: Current Knowledge, Challenges, and Future Perspectives

by Tindaro Bongiovanni, Mathieu Lacome, Vassilios Fanos, Giulia Martera, Erika Cione and Roberto Cannataro

Proteomes 2022, 10(3), 27; https://doi.org/10.3390/proteomes10030027 - 10 Aug 2022

Cited by 6 | Viewed by 2782

Abstract

Metabolomics is a promising tool for studying exercise physiology and exercise-associated metabolism. It has recently been defined with the term “sportomics” due to metabolomics’ capability to characterize several metabolites in several biological samples simultaneously. This narrative review on exercise metabolomics provides an initial [...] Read more.

Metabolomics is a promising tool for studying exercise physiology and exercise-associated metabolism. It has recently been defined with the term “sportomics” due to metabolomics’ capability to characterize several metabolites in several biological samples simultaneously. This narrative review on exercise metabolomics provides an initial and brief overview of the different metabolomics technologies, sample collection, and further processing steps employed for sport. It also discusses the data analysis and its biological interpretation. Thus, we do not cover sample collection, preparation, and analysis paragraphs in detail here but outline a general outlook to help the reader to understand the metabolomics studies conducted in team-sports athletes, alongside endeavoring to recognize existing or emergent trends and deal with upcoming directions in the field of exercise metabolomics in a team-sports setting. Full article

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32 pages, 950 KiB

Open AccessReview

Proteomic Discovery and Validation of Novel Fluid Biomarkers for Improved Patient Selection and Prediction of Clinical Outcomes in Alzheimer’s Disease Patient Cohorts

by Shivangi Awasthi, Daniel S. Spellman and Nathan G. Hatcher

Proteomes 2022, 10(3), 26; https://doi.org/10.3390/proteomes10030026 - 01 Aug 2022

Cited by 7 | Viewed by 4337

Abstract

Alzheimer’s disease (AD) is an irreversible neurodegenerative disease characterized by progressive cognitive decline. The two cardinal neuropathological hallmarks of AD include the buildup of cerebral β amyloid (Aβ) plaques and neurofibrillary tangles of hyperphosphorylated tau. The current disease-modifying treatments are still not effective [...] Read more.

Alzheimer’s disease (AD) is an irreversible neurodegenerative disease characterized by progressive cognitive decline. The two cardinal neuropathological hallmarks of AD include the buildup of cerebral β amyloid (Aβ) plaques and neurofibrillary tangles of hyperphosphorylated tau. The current disease-modifying treatments are still not effective enough to lower the rate of cognitive decline. There is an urgent need to identify early detection and disease progression biomarkers that can facilitate AD drug development. The current established readouts based on the expression levels of amyloid beta, tau, and phospho-tau have shown many discrepancies in patient samples when linked to disease progression. There is an urgent need to identify diagnostic and disease progression biomarkers from blood, cerebrospinal fluid (CSF), or other biofluids that can facilitate the early detection of the disease and provide pharmacodynamic readouts for new drugs being tested in clinical trials. Advances in proteomic approaches using state-of-the-art mass spectrometry are now being increasingly applied to study AD disease mechanisms and identify drug targets and novel disease biomarkers. In this report, we describe the application of quantitative proteomic approaches for understanding AD pathophysiology, summarize the current knowledge gained from proteomic investigations of AD, and discuss the development and validation of new predictive and diagnostic disease biomarkers. Full article

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

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14 pages, 1430 KiB

Open AccessArticle

High-Resolution Secretome Analysis of Chemical Hypoxia Treated Cells Identifies Putative Biomarkers of Chondrosarcoma

by Donatella Pia Spanò, Simone Bonelli, Matteo Calligaris, Anna Paola Carreca, Claudia Carcione, Giovanni Zito, Aldo Nicosia, Sergio Rizzo and Simone Dario Scilabra

Proteomes 2022, 10(3), 25; https://doi.org/10.3390/proteomes10030025 - 20 Jul 2022

Cited by 4 | Viewed by 2653

Abstract

Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic process of chondrosarcoma. As a consequence, there are no approved therapies for [...] Read more.

Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic process of chondrosarcoma. As a consequence, there are no approved therapies for this tumor and surgical resection is the only treatment currently available. Moreover, there are no available biomarkers for this type of tumor, and chondrosarcoma classification relies on operator-dependent histopathological assessment. Reliable biomarkers of chondrosarcoma are urgently needed, as well as greater understanding of the molecular mechanisms of its development for translational purposes. Hypoxia is a central feature of chondrosarcoma progression. The hypoxic tumor microenvironment of chondrosarcoma triggers a number of cellular events, culminating in increased invasiveness and migratory capability. Herein, we analyzed the effects of chemically-induced hypoxia on the secretome of SW 1353, a human chondrosarcoma cell line, using high-resolution quantitative proteomics. We found that hypoxia induced unconventional protein secretion and the release of proteins associated to exosomes. Among these proteins, which may be used to monitor chondrosarcoma development, we validated the increased secretion in response to hypoxia of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme well-known for its different functional roles in a wide range of tumors. In conclusion, by analyzing the changes induced by hypoxia in the secretome of chondrosarcoma cells, we identified molecular mechanisms that can play a role in chondrosarcoma progression and pinpointed proteins, including GAPDH, that may be developed as potential biomarkers for the diagnosis and therapeutic management of chondrosarcoma. Full article

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17 pages, 610 KiB

Open AccessReview

Proteomics for Biomarker Discovery for Diagnosis and Prognosis of Kidney Transplantation Rejection

by Luís M. Ramalhete, Rúben Araújo, Aníbal Ferreira and Cecília R. C. Calado

Proteomes 2022, 10(3), 24; https://doi.org/10.3390/proteomes10030024 - 02 Jul 2022

Cited by 8 | Viewed by 3293

Abstract

Renal transplantation is currently the treatment of choice for end-stage kidney disease, enabling a quality of life superior to dialysis. Despite this, all transplanted patients are at risk of allograft rejection processes. The gold-standard diagnosis of graft rejection, based on histological analysis of [...] Read more.

Renal transplantation is currently the treatment of choice for end-stage kidney disease, enabling a quality of life superior to dialysis. Despite this, all transplanted patients are at risk of allograft rejection processes. The gold-standard diagnosis of graft rejection, based on histological analysis of kidney biopsy, is prone to sampling errors and carries high costs and risks associated with such invasive procedures. Furthermore, the routine clinical monitoring, based on urine volume, proteinuria, and serum creatinine, usually only detects alterations after graft histologic damage and does not differentiate between the diverse etiologies. Therefore, there is an urgent need for new biomarkers enabling to predict, with high sensitivity and specificity, the rejection processes and the underlying mechanisms obtained from minimally invasive procedures to be implemented in routine clinical surveillance. These new biomarkers should also detect the rejection processes as early as possible, ideally before the 78 clinical outputs, while enabling balanced immunotherapy in order to minimize rejections and reducing the high toxicities associated with these drugs. Proteomics of biofluids, collected through non-invasive or minimally invasive analysis, e.g., blood or urine, present inherent characteristics that may provide biomarker candidates. The current manuscript reviews biofluids proteomics toward biomarkers discovery that specifically identify subclinical, acute, and chronic immune rejection processes while allowing for the discrimination between cell-mediated or antibody-mediated processes. In time, these biomarkers will lead to patient risk stratification, monitoring, and personalized and more efficient immunotherapies toward higher graft survival and patient quality of life. Full article

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

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13 pages, 3760 KiB

Open AccessArticle

Integrative Proteomic and Phosphoproteomic Analyses of Hypoxia-Treated Pulmonary Artery Smooth Muscle Cells

by Ang Luo, Rongrong Hao, Xia Zhou, Yangfan Jia and Haiyang Tang

Proteomes 2022, 10(3), 23; https://doi.org/10.3390/proteomes10030023 - 28 Jun 2022

Cited by 2 | Viewed by 3010

Abstract

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the main causes of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Hypoxia is an important factor related to PAH and can induce the excessive proliferation of PASMCs and inhibit apoptosis. [...] Read more.

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the main causes of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Hypoxia is an important factor related to PAH and can induce the excessive proliferation of PASMCs and inhibit apoptosis. To explore the possible mechanism of hypoxia-related PAH, human PASMCs are exposed to hypoxia for 24 h and tandem mass tag (TMT)-based quantitative proteomic and phosphoproteomic analyses are performed. Proteomic analysis revealed 134 proteins are significantly changed (p < 0.05, |log2 (fold change)| > log2 [1.1]), of which 48 proteins are upregulated and 86 are downregulated. Some of the changed proteins are verified by using qRT-PCR and Western blotting. Phosphoproteomic analysis identified 404 significantly changed (p < 0.05, |log2 (fold change)| > log2 [1.1]) phosphopeptides. Among them, 146 peptides are upregulated while 258 ones are downregulated. The kinase-substrate enrichment analysis revealed kinases such as P21 protein-activated kinase 1/2/4 (PAK1/2/4), protein-kinase cGMP-dependent 1 and 2 (PRKG1/2), and mitogen-activated protein-kinase 4/6/7 (MAP2K4/6/7) are significantly enriched and activated. For all the significantly changed proteins or phosphoproteins, a comprehensive pathway analysis is performed. In general, this study furthers our understanding of the mechanism of hypoxia-induced PAH. Full article

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Proteomes, Volume 10, Issue 3 (September 2022) – 11 articles

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