scholarly journals Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications

2015 ◽  
Vol 14 (3) ◽  
pp. 609-620 ◽  
Author(s):  
Jana Paulech ◽  
Kiersten A. Liddy ◽  
Kasper Engholm-Keller ◽  
Melanie Y. White ◽  
Stuart J. Cordwell
Keyword(s):  
Sulfonic Acid ◽  
Global Analysis ◽  
Post Translational Modifications

scholarly journals Enabling global analysis of protein citrullination and homocitrullination via biotin thiol tag-assisted mass spectrometry

2021 ◽  
Author(s):  
Lingjun Li ◽  
Yatao Shi ◽  
Zihui Li ◽  
Bin Wang ◽  
Xudong Shi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Global Analysis ◽  
Protein Structures ◽  
Isotopic Labeling ◽  
Underlying Mechanism ◽  
Biological Processes ◽  
Disease Pathogenesis ◽  
Post Translational Modifications ◽  
Mouse Tissues ◽  
Global Mapping

Abstract Citrullination and homocitrullination are key post-translational modifications (PTMs) that affect protein structures and functions. Although they have been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize these PTMs. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of these two PTMs simultaneously via mass spectrometry. We perform global mapping of the citrullination and homocitrullination proteomes of mouse tissues. In total, we identify 1,198 citrullination sites and 108 homocitrullination sites from 619 and 79 proteins, respectively, representing the largest datasets to date. We discover novel distribution and functions of these two PTMs. We also perform multiplexing quantitative analysis via isotopic labeling techniques. This study depicts a landscape of protein citrullination and homocitrullination and lays the foundation to further decipher their physiological and pathological roles.

scholarly journals Enabling global analysis of protein citrullination and homocitrullination via biotin thiol tag-assisted mass spectrometry

2022 ◽  
Author(s):  
Lingjun Li ◽  
Yatao Shi ◽  
Zihui Li ◽  
Bin Wang ◽  
Xudong Shi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Global Analysis ◽  
Protein Structures ◽  
Isotopic Labeling ◽  
Underlying Mechanism ◽  
Biological Processes ◽  
Disease Pathogenesis ◽  
Post Translational Modifications ◽  
Mouse Tissues ◽  
Global Mapping

Abstract Citrullination and homocitrullination are key post-translational modifications (PTMs) that affect protein structures and functions. Although they have been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize these PTMs. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of these two PTMs simultaneously via mass spectrometry. We perform global mapping of the citrullination and homocitrullination proteomes of mouse tissues. In total, we identify 691 citrullination sites and 81 homocitrullination sites from 432 and 63 proteins, respectively, representing the largest datasets to date. We discover novel distribution and functions of these two PTMs. We also perform multiplexing quantitative analysis via isotopic labeling techniques. This study depicts a landscape of protein citrullination and homocitrullination and lays the foundation for further deciphering their physiological and pathological roles.

scholarly journals A global analysis of low-complexity regions in the Trypanosoma brucei proteome reveals enrichment in the C-terminus of nucleic acid binding proteins providing potential targets of phosphorylation

2020 ◽  
Vol 5 ◽  
pp. 219
Author(s):  
Mathieu Cayla ◽  
Keith R. Matthews ◽  
Alasdair C. Ivens
Keyword(s):  
Gene Regulation ◽  
Nucleic Acid ◽  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Global Analysis ◽  
Low Complexity ◽  
Nucleic Acid Binding ◽  
Post Translational Modifications ◽  
Gene Expression Control ◽  
Nucleic Acid Binding Proteins

Background: Low-complexity regions (LCRs) on proteins have attracted increasing attention recently due to their role in the assembly of membraneless organelles or granules by liquid-liquid phase separation. Several examples of such granules have been shown to sequester RNA and proteins in an inactive state, providing an important mechanism for dynamic post-transcriptional gene regulation. In trypanosome parasites, post-transcriptional control overwhelmingly dominates gene regulation due to the organisation of their genome into polycistronic transcription units. The purpose of the current study was to generate a substantially more comprehensive genome-wide survey of LCRs on trypanosome proteins than currently available . Methods: Using the Shannon’s entropy method, provided in the R package ‘entropy’, we identified LCRs in the proteome of Trypanosoma brucei. Our analysis predicts LCRs and their positional enrichment in distinct protein cohorts and superimposes on this a range of post-translational modifications derived from available experimental datasets. Results: We have identified 8162 LCRs present on 4914 proteins, representing 42% of the proteome, placing Trypanosoma brucei among the eukaryotes with the highest percentage of LCRs. Our results highlight the enrichment of LCRs in the C-terminal region of predicted nucleic acid binding proteins, these acting as favoured sites for potential phosphorylation. Phosphorylation represents 51% of the post-translational modifications present on LCRs compared to 16% on the rest of the proteome. Conclusions: The post-translational modifications of LCRs, and in particular phosphorylation events, could contribute to post-transcriptional gene expression control and the dynamics of protein targeting to membraneless organelles in kinetoplastid parasites.

scholarly journals Global proteomic profiling of primary macrophages during M. tuberculosis infection identifies TAX1BP1 as a mediator of autophagy targeting

2019 ◽  
Author(s):  
Jonathan M. Budzik ◽  
Nick E. Garelis ◽  
Teresa Repasy ◽  
Allison W. Roberts ◽  
Lauren M. Popov ◽  
...  
Keyword(s):  
Global Analysis ◽  
Nucleocytoplasmic Transport ◽  
Host Protein ◽  
Protein Abundance ◽  
Proteomic Profiling ◽  
Cell Fates ◽  
Post Translational Modifications ◽  
Functional Capabilities ◽  
Mrna Metabolism ◽  
Adaptor Recruitment

AbstractMacrophages are highly plastic cells that adopt diverse functional capabilities and play critical roles in immunity, cancer, and tissue homeostasis, but how these different cell fates and activities are triggered in response to their environmental cues is not well understood. We used new proteomic tools to identify protein post-translational modifications (PTMs) that control antibacterial responses of macrophages. Here, we report an unbiased and global analysis of the changes in host protein abundance, phosphorylation, and ubiquitylation, during the first 24-hours of Mycobacterium tuberculosis (Mtb) infection of primary macrophages. We discovered 1379 proteins with changes in their phosphorylation state and 591 proteins with changes in their ubiquitylation in response to Mtb infection. We identified pathways regulated by phosphorylation and ubiquitylation that weren’t reflected by changes in protein abundance, indicating that the activity of these pathways was regulated. These include pathways known to be regulated by ubiquitylation and phosphorylation (e.g. autophagy) as well as pathways that were not known to be regulated during Mtb infection (e.g. nucleocytoplasmic transport and mRNA metabolism). We identified an enrichment in phosphorylation of autophagy receptors (TAX1BP1, p62, optineurin, BNIP3L), several of which were not previously implicated in the host response to Mtb infection. We found that p62 deficiency blocks ubiquitylation and TAX1BP1 deficiency enhances ubiquitylation, suggesting p62 ubiquitylation acts as an amplification loop by promoting downstream adaptor recruitment and serves as a platform for recruitment of ubiquitin. Our results show that TAX1BP1 mediates clearance of ubiquitylated Mtb and targets the bacteria to LC3-positive phagophores. Taken together, our proteomic profiling is likely a valuable resource for initiating mechanistic studies of macrophage biology.

scholarly journals A global analysis of low-complexity regions in the Trypanosoma brucei proteome reveals enrichment in the C-terminus of nucleic acid binding proteins providing potential targets of phosphorylation

2020 ◽  
Vol 5 ◽  
pp. 219
Author(s):  
Mathieu Cayla ◽  
Keith R. Matthews ◽  
Alasdair C. Ivens
Keyword(s):  
Gene Regulation ◽  
Nucleic Acid ◽  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Global Analysis ◽  
Low Complexity ◽  
Nucleic Acid Binding ◽  
Post Translational Modifications ◽  
Gene Expression Control ◽  
Nucleic Acid Binding Proteins

Background: Low-complexity regions (LCRs) on proteins have attracted increasing attention recently due to their role in the assembly of membraneless organelles or granules by liquid-liquid phase separation. Several examples of such granules have been shown to sequester RNA and proteins in an inactive state, providing an important mechanism for dynamic post-transcriptional gene regulation. In trypanosome parasites, post-transcriptional control overwhelmingly dominates gene regulation due to the organisation of their genome into polycistronic transcription units. The purpose of the current study was to generate a substantially more comprehensive genome-wide survey of LCRs on trypanosome proteins than currently available. Methods: Using the Shannon’s entropy method, provided in the R package ‘entropy’, we identified LCRs in the proteome of Trypanosoma brucei. Our analysis predicts LCRs and their positional enrichment in distinct protein cohorts and superimposes on this a range of post-translational modifications derived from available experimental datasets. Results: Our results highlight the enrichment of LCRs in the C-terminal region of predicted nucleic acid binding proteins, these acting as favoured sites for potential phosphorylation. Conclusions: The post-translational modifications of LCRs, and in particular the phosphorylation events, could contribute to post-transcriptional gene expression control and the dynamics of protein targeting to membraneless organelles in kinetoplastid parasites.

Electron Microscopy of Mycoplasma Pneumoniae

1971 ◽  
Vol 29 ◽  
pp. 258-259 ◽  
Author(s):  
E. S. Boatman ◽  
G. E. Kenny
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Growth Phase ◽  
Hela Cells ◽  
Sulfonic Acid ◽  
Gamma Globulin ◽  
Horse Serum ◽  
Morphological Aspects ◽  
The Family

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.

Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins

2020 ◽  
Vol 477 (7) ◽  
pp. 1219-1225 ◽  
Author(s):  
Nikolai N. Sluchanko
Keyword(s):  
Protein Function ◽  
Intrinsically Disordered Protein ◽  
Structural Basis ◽  
Major Protein ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Intrinsically Disordered ◽  
Biochemical Journal ◽  
Multiple Binding ◽  
And Control

Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

Dicarbonyl derived post-translational modifications: chemistry bridging biology and aging-related disease

2020 ◽  
Vol 64 (1) ◽  
pp. 97-110
Author(s):  
Christian Sibbersen ◽  
Mogens Johannsen
Keyword(s):  
Mass Spectrometry ◽  
Future Research ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Dicarbonyl Compounds ◽  
Protein Targets ◽  
Post Translational Modifications ◽  
Reactive Protein ◽  
Glycation End Products ◽  
Direct Mass Spectrometry

Abstract In living systems, nucleophilic amino acid residues are prone to non-enzymatic post-translational modification by electrophiles. α-Dicarbonyl compounds are a special type of electrophiles that can react irreversibly with lysine, arginine, and cysteine residues via complex mechanisms to form post-translational modifications known as advanced glycation end-products (AGEs). Glyoxal, methylglyoxal, and 3-deoxyglucosone are the major endogenous dicarbonyls, with methylglyoxal being the most well-studied. There are several routes that lead to the formation of dicarbonyl compounds, most originating from glucose and glucose metabolism, such as the non-enzymatic decomposition of glycolytic intermediates and fructosyl amines. Although dicarbonyls are removed continuously mainly via the glyoxalase system, several conditions lead to an increase in dicarbonyl concentration and thereby AGE formation. AGEs have been implicated in diabetes and aging-related diseases, and for this reason the elucidation of their structure as well as protein targets is of great interest. Though the dicarbonyls and reactive protein side chains are of relatively simple nature, the structures of the adducts as well as their mechanism of formation are not that trivial. Furthermore, detection of sites of modification can be demanding and current best practices rely on either direct mass spectrometry or various methods of enrichment based on antibodies or click chemistry followed by mass spectrometry. Future research into the structure of these adducts and protein targets of dicarbonyl compounds may improve the understanding of how the mechanisms of diabetes and aging-related physiological damage occur.

The challenge of detecting modifications on proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 135-153 ◽  
Author(s):  
Lauren Elizabeth Smith ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Chemical Properties ◽  
Lysine Acetylation ◽  
Mass Determination ◽  
Post Translational Modifications ◽  
Site Specific ◽  
The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

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