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15 pages, 4551 KiB

Open AccessArticle

A Comprehensive Study of Gradient Conditions for Deep Proteome Discovery in a Complex Protein Matrix

by Xing Wei, Pei N. Liu, Brian P. Mooney, Thao Thi Nguyen and C. Michael Greenlief

Int. J. Mol. Sci. 2022, 23(19), 11714; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231911714 - 03 Oct 2022

Cited by 1 | Viewed by 1739

Bottom–up mass-spectrometry-based proteomics is a well-developed technology based on complex peptide mixtures from proteolytic cleavage of proteins and is widely applied in protein identification, characterization, and quantitation. A tims-ToF mass spectrometer is an excellent platform for bottom–up proteomics studies due to its rapid [...] Read more.

Bottom–up mass-spectrometry-based proteomics is a well-developed technology based on complex peptide mixtures from proteolytic cleavage of proteins and is widely applied in protein identification, characterization, and quantitation. A tims-ToF mass spectrometer is an excellent platform for bottom–up proteomics studies due to its rapid acquisition with high sensitivity. It remains challenging for bottom–up proteomics approaches to achieve 100% proteome coverage. Liquid chromatography (LC) is commonly used prior to mass spectrometry (MS) analysis to fractionate peptide mixtures, and the LC gradient can affect the peptide fractionation and proteome coverage. We investigated the effects of gradient type and time duration to find optimal gradient conditions. Five gradient types (linear, logarithm-like, exponent-like, stepwise, and step-linear), three different gradient lengths (22 min, 44 min, and 66 min), two sample loading amounts (100 ng and 200 ng), and two loading conditions (the use of trap column and no trap column) were studied. The effect of these chromatography variables on protein groups, peptides, and spectral counts using HeLa cell digests was explored. The results indicate that (1) a step-linear gradient performs best among the five gradient types studied; (2) the optimal gradient duration depends on protein sample loading amount; (3) the use of a trap column helps to enhance protein identification, especially low-abundance proteins; (4) MSFragger and PEAKS Studio have high similarity in protein group identification; (5) MSFragger identified more protein groups among the different gradient conditions compared to PEAKS Studio; and (6) combining results from both database search engines can expand identified protein groups by 9–11%. Full article

(This article belongs to the Special Issue Biological Systems at the Protein Level 2.0)

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25 pages, 5677 KiB

Open AccessArticle

Molecular Framework of Mouse Endothelial Cell Dysfunction during Inflammation: A Proteomics Approach

by Michael T. Rossi, Jordan C. Langston, Narender Singh, Carmen Merali, Qingliang Yang, Salim Merali, Balabhaskar Prabhakarpandian, Laurie E. Kilpatrick and Mohammad F. Kiani

Int. J. Mol. Sci. 2022, 23(15), 8399; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23158399 - 29 Jul 2022

Cited by 4 | Viewed by 1906

Abstract

A key aspect of cytokine-induced changes as observed in sepsis is the dysregulated activation of endothelial cells (ECs), initiating a cascade of inflammatory signaling leading to leukocyte adhesion/migration and organ damage. The therapeutic targeting of ECs has been hampered by concerns regarding organ-specific [...] Read more.

A key aspect of cytokine-induced changes as observed in sepsis is the dysregulated activation of endothelial cells (ECs), initiating a cascade of inflammatory signaling leading to leukocyte adhesion/migration and organ damage. The therapeutic targeting of ECs has been hampered by concerns regarding organ-specific EC heterogeneity and their response to inflammation. Using in vitro and in silico analysis, we present a comprehensive analysis of the proteomic changes in mouse lung, liver and kidney ECs following exposure to a clinically relevant cocktail of proinflammatory cytokines. Mouse lung, liver and kidney ECs were incubated with TNF-α/IL-1β/IFN-γ for 4 or 24 h to model the cytokine-induced changes. Quantitative label-free global proteomics and bioinformatic analysis performed on the ECs provide a molecular framework for the EC response to inflammatory stimuli over time and organ-specific differences. Gene Ontology and PANTHER analysis suggest why some organs are more susceptible to inflammation early on, and show that, as inflammation progresses, some protein expression patterns become more uniform while additional organ-specific proteins are expressed. These findings provide an in-depth understanding of the molecular changes involved in the EC response to inflammation and can support the development of drugs targeting ECs within different organs. Data are available via ProteomeXchange (identifier PXD031804). Full article

(This article belongs to the Special Issue Biological Systems at the Protein Level 2.0)

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21 pages, 2588 KiB

Open AccessArticle

Protein Expression of AEBP1, MCM4, and FABP4 Differentiate Osteogenic, Adipogenic, and Mesenchymal Stromal Stem Cells

by Thorben Sauer, Giulia Facchinetti, Michael Kohl, Justyna M. Kowal, Svitlana Rozanova, Julia Horn, Hagen Schmal, Ivo Kwee, Arndt-Peter Schulz, Sonja Hartwig, Moustapha Kassem, Jens K. Habermann and Timo Gemoll

Int. J. Mol. Sci. 2022, 23(5), 2568; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23052568 - 25 Feb 2022

Cited by 5 | Viewed by 2788

Abstract

Mesenchymal stem cells (MSCs) gain an increasing focus in the field of regenerative medicine due to their differentiation abilities into chondrocytes, adipocytes, and osteoblastic cells. However, it is apparent that the transformation processes are extremely complex and cause cellular heterogeneity. The study aimed [...] Read more.

Mesenchymal stem cells (MSCs) gain an increasing focus in the field of regenerative medicine due to their differentiation abilities into chondrocytes, adipocytes, and osteoblastic cells. However, it is apparent that the transformation processes are extremely complex and cause cellular heterogeneity. The study aimed to characterize differences between MSCs and cells after adipogenic (AD) or osteoblastic (OB) differentiation at the proteome level. Comparative proteomic profiling was performed using tandem mass spectrometry in data-independent acquisition mode. Proteins were quantified by deep neural networks in library-free mode and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. We analyzed 4108 proteins across all samples, which revealed a distinct clustering between MSCs and cell differentiation states. Protein expression profiling identified activation of the Peroxisome proliferator-activated receptors (PPARs) signaling pathway after AD. In addition, two distinct protein marker panels could be defined for osteoblastic and adipocytic cell lineages. Hereby, overexpression of AEBP1 and MCM4 for OB as well as of FABP4 for AD was detected as the most promising molecular markers. Combination of deep neural network and machine-learning algorithms with data-independent mass spectrometry distinguish MSCs and cell lineages after adipogenic or osteoblastic differentiation. We identified specific proteins as the molecular basis for bone formation, which could be used for regenerative medicine in the future. Full article

(This article belongs to the Special Issue Biological Systems at the Protein Level 2.0)

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22 pages, 3319 KiB

Open AccessArticle

Biochemical Mapping of Pyrodinium bahamense Unveils Molecular Underpinnings behind Organismal Processes

by Bryan John J. Subong, Zabrina Bernice L. Malto, Arturo O. Lluisma, Rhodora V. Azanza and Lilibeth A. Salvador-Reyes

Int. J. Mol. Sci. 2021, 22(24), 13332; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222413332 - 11 Dec 2021

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Abstract

Proteins, lipids, and carbohydrates from the harmful algal bloom (HAB)-causing organism Pyrodinium bahamense were characterized to obtain insights into the biochemical processes in this environmentally relevant dinoflagellate. Shotgun proteomics using label-free quantitation followed by proteome mapping using the P. bahamense transcriptome and translated [...] Read more.

Proteins, lipids, and carbohydrates from the harmful algal bloom (HAB)-causing organism Pyrodinium bahamense were characterized to obtain insights into the biochemical processes in this environmentally relevant dinoflagellate. Shotgun proteomics using label-free quantitation followed by proteome mapping using the P. bahamense transcriptome and translated protein databases of Marinovum algicola, Alexandrium sp., Cylindrospermopsis raciborskii, and Symbiodinium kawagutii for annotation enabled the characterization of the proteins in P. bahamense. The highest number of annotated hits were obtained from M. algicola and highlighted the contribution of microorganisms associated with P. bahamense. Proteins involved in dimethylsulfoniopropionate (DMSP) degradation such as propionyl CoA synthethase and acryloyl-CoA reductase were identified, suggesting the DMSP cleavage pathway as the preferred route in this dinoflagellate. Most of the annotated proteins were involved in amino acid biosynthesis and carbohydrate degradation and metabolism, indicating the active roles of these molecules in the vegetative stage of P. bahamense. This characterization provides baseline information on the cellular machinery and the molecular basis of the ecophysiology of P. bahamense. Full article

(This article belongs to the Special Issue Biological Systems at the Protein Level 2.0)

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19 pages, 16487 KiB

Open AccessArticle

Serum Metabolomic and Lipidomic Profiling Reveals Novel Biomarkers of Efficacy for Benfotiamine in Alzheimer’s Disease

by Ruchika Bhawal, Qin Fu, Elizabeth T. Anderson, Gary E. Gibson and Sheng Zhang

Int. J. Mol. Sci. 2021, 22(24), 13188; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222413188 - 07 Dec 2021

Cited by 13 | Viewed by 5189

Abstract

Serum metabolomics and lipidomics are powerful approaches for discovering unique biomarkers in various diseases and associated therapeutics and for revealing metabolic mechanisms of both. Treatment with Benfotiamine (BFT), a thiamine prodrug, for one year produced encouraging results for patients with mild cognitive impairment [...] Read more.

Serum metabolomics and lipidomics are powerful approaches for discovering unique biomarkers in various diseases and associated therapeutics and for revealing metabolic mechanisms of both. Treatment with Benfotiamine (BFT), a thiamine prodrug, for one year produced encouraging results for patients with mild cognitive impairment and mild Alzheimer’s disease (AD). In this study, a parallel metabolomics and lipidomics approach was applied for the first exploratory investigation on the serum metabolome and lipidome of patients treated with BFT. A total of 315 unique metabolites and 417 lipids species were confidently identified and relatively quantified. Rigorous statistical analyses revealed significant differences between the placebo and BFT treatment groups in 25 metabolites, including thiamine, tyrosine, tryptophan, lysine, and 22 lipid species, mostly belonging to phosphatidylcholines. Additionally, 10 of 11 metabolites and 14 of 15 lipid species reported in previous literature to follow AD progression changed in the opposite direction to those reported to reflect AD progression. Enrichment and pathway analyses show that significantly altered metabolites by BFT are involved in glucose metabolism and biosynthesis of aromatic amino acids. Our study discovered that multiple novel biomarkers and multiple mechanisms that may underlie the benefit of BFT are potential therapeutic targets in AD and should be validated in studies with larger sample sizes. Full article

(This article belongs to the Special Issue Biological Systems at the Protein Level 2.0)

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26 pages, 3619 KiB

Open AccessArticle

Chronic Activation of AMPK Induces Mitochondrial Biogenesis through Differential Phosphorylation and Abundance of Mitochondrial Proteins in Dictyostelium discoideum

by Malgorzata Heidorn-Czarna, Herbert-Michael Heidorn, Sanjanie Fernando, Oana Sanislav, Wieslawa Jarmuszkiewicz, Rupert Mutzel and Paul R. Fisher

Int. J. Mol. Sci. 2021, 22(21), 11675; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222111675 - 28 Oct 2021

Cited by 2 | Viewed by 3282

Abstract

Mitochondrial biogenesis is a highly controlled process that depends on diverse signalling pathways responding to cellular and environmental signals. AMP-activated protein kinase (AMPK) is a critical metabolic enzyme that acts at a central control point in cellular energy homeostasis. Numerous studies have revealed [...] Read more.

Mitochondrial biogenesis is a highly controlled process that depends on diverse signalling pathways responding to cellular and environmental signals. AMP-activated protein kinase (AMPK) is a critical metabolic enzyme that acts at a central control point in cellular energy homeostasis. Numerous studies have revealed the crucial roles of AMPK in the regulation of mitochondrial biogenesis; however, molecular mechanisms underlying this process are still largely unknown. Previously, we have shown that, in cellular slime mould Dictyostelium discoideum, the overexpression of the catalytic α subunit of AMPK led to enhanced mitochondrial biogenesis, which was accompanied by reduced cell growth and aberrant development. Here, we applied mass spectrometry-based proteomics of Dictyostelium mitochondria to determine the impact of chronically active AMPKα on the phosphorylation state and abundance of mitochondrial proteins and to identify potential protein targets leading to the biogenesis of mitochondria. Our results demonstrate that enhanced mitochondrial biogenesis is associated with variations in the phosphorylation levels and abundance of proteins related to energy metabolism, protein synthesis, transport, inner membrane biogenesis, and cellular signalling. The observed changes are accompanied by elevated mitochondrial respiratory activity in the AMPK overexpression strain. Our work is the first study reporting on the global phosphoproteome profiling of D. discoideum mitochondria and its changes as a response to constitutively active AMPK. We also propose an interplay between the AMPK and mTORC1 signalling pathways in controlling the cellular growth and biogenesis of mitochondria in Dictyostelium as a model organism. Full article

(This article belongs to the Special Issue Biological Systems at the Protein Level 2.0)

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Review

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35 pages, 1450 KiB

Open AccessReview

Improving Protein Quantity and Quality—The Next Level of Plant Molecular Farming

by Hai Liu and Michael P. Timko

Int. J. Mol. Sci. 2022, 23(3), 1326; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031326 - 25 Jan 2022

Cited by 13 | Viewed by 5157

Abstract

Plants offer several unique advantages in the production of recombinant pharmaceuticals for humans and animals. Although numerous recombinant proteins have been expressed in plants, only a small fraction have been successfully put into use. The hugely distinct expression systems between plant and animal [...] Read more.

Plants offer several unique advantages in the production of recombinant pharmaceuticals for humans and animals. Although numerous recombinant proteins have been expressed in plants, only a small fraction have been successfully put into use. The hugely distinct expression systems between plant and animal cells frequently cause insufficient yield of the recombinant proteins with poor or undesired activity. To overcome the issues that greatly constrain the development of plant-produced pharmaceuticals, great efforts have been made to improve expression systems and develop alternative strategies to increase both the quantity and quality of the recombinant proteins. Recent technological revolutions, such as targeted genome editing, deconstructed vectors, virus-like particles, and humanized glycosylation, have led to great advances in plant molecular farming to meet the industrial manufacturing and clinical application standards. In this review, we discuss the technological advances made in various plant expression platforms, with special focus on the upstream designs and milestone achievements in improving the yield and glycosylation of the plant-produced pharmaceutical proteins. Full article

(This article belongs to the Special Issue Biological Systems at the Protein Level 2.0)

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25 pages, 2222 KiB

Open AccessReview

Short Peptides Make a Big Difference: The Role of Botany-Derived AMPs in Disease Control and Protection of Human Health

by Xiumei Luo, Wenxian Wu, Li Feng, Haim Treves and Maozhi Ren

Int. J. Mol. Sci. 2021, 22(21), 11363; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222111363 - 21 Oct 2021

Cited by 6 | Viewed by 2270

Abstract

Botany-derived antimicrobial peptides (BAMPs), a class of small, cysteine-rich peptides produced in plants, are an important component of the plant immune system. Both in vivo and in vitro experiments have demonstrated their powerful antimicrobial activity. Besides in plants, BAMPs have cross-kingdom applications in [...] Read more.

Botany-derived antimicrobial peptides (BAMPs), a class of small, cysteine-rich peptides produced in plants, are an important component of the plant immune system. Both in vivo and in vitro experiments have demonstrated their powerful antimicrobial activity. Besides in plants, BAMPs have cross-kingdom applications in human health, with toxic and/or inhibitory effects against a variety of tumor cells and viruses. With their diverse molecular structures, broad-spectrum antimicrobial activity, multiple mechanisms of action, and low cytotoxicity, BAMPs provide ideal backbones for drug design, and are potential candidates for plant protection and disease treatment. Lots of original research has elucidated the properties and antimicrobial mechanisms of BAMPs, and characterized their surface receptors and in vivo targets in pathogens. In this paper, we review and introduce five kinds of representative BAMPs belonging to the pathogenesis-related protein family, dissect their antifungal, antiviral, and anticancer mechanisms, and forecast their prospects in agriculture and global human health. Through the deeper understanding of BAMPs, we provide novel insights for their applications in broad-spectrum and durable plant disease prevention and control, and an outlook on the use of BAMPs in anticancer and antiviral drug design. Full article

(This article belongs to the Special Issue Biological Systems at the Protein Level 2.0)

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