MS-based quantification of RhoA/B and RhoC ADP-ribosylation

Schröder, Arne; Benski, Anastasia; Oltmanns, Anne; Just, Ingo; Rohrbeck, Astrid; Pich, Andreas

doi:10.1016/j.jchromb.2018.06.007

Cited by 3 publications

(3 citation statements)

References 17 publications

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“…While general actin ribosylation by CDT has already been shown ( Gülke et al, 2001 ), here we could confirm for the first time that CDT ribosylates actin specifically on Arg-177. Interestingly, HCD fragmentation produces no fragment ion containing the ribosylation site on Arg-177, despite detecting the mono-ADP-ribose fragment at 348.2 m/z ( Figure 6 ), described as a diagnostic ADP ribosylation marker ( Schröder et al, 2018 ). However, the CID fragmentation method produced fragment ions containing the modification and served as an additional verification on the localization of the ADP ribosylation ( Supplementary Figure 2 ).…”

Section: Discussionmentioning

confidence: 99%

The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells

2021

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Clostridioides difficile is a major cause of nosocomial infection worldwide causing antibiotic-associated diarrhea and some cases are leading to pseudomembranous colitis. The main virulence factors are toxin A and toxin B. Hypervirulent strains of C. difficile are linked to higher mortality rates and most of these strains produce additionally the C. difficile binary toxin (CDT) that possesses two subunits, CDTa and CDTb. The latter is responsible for binding and transfer of CDTa into the cytoplasm of target cells; CDTa is an ADP ribosyltransferase catalyzing the modification of actin fibers that disturbs the actin vs microtubule balance and induces microtubule-based protrusions of the cell membrane increasing the adherence of C. difficile. The underlying mechanisms remain elusive. Thus, we performed a screening experiment using MS-based proteomics and phosphoproteomics techniques. Epithelial Hep-2 cells were treated with CDTa and CDTb in a multiplexed study for 4 and 8 h. Phosphopeptide enrichment was performed using affinity chromatography with TiO2 and Fe-NTA; for quantification, a TMT-based approach and DDA measurements were used. More than 4,300 proteins and 5,600 phosphosites were identified and quantified at all time points. Although only moderate changes were observed on proteome level, the phosphorylation level of nearly 1,100 phosphosites responded to toxin treatment. The data suggested that CSNK2A1 might act as an effector kinase after treatment with CDT. Additionally, we confirmed ADP-ribosylation on Arg-177 of actin and the kinetic of this modification for the first time.

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Section: Discussionmentioning

confidence: 99%

The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells

2021

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“…The small GTPases RhoA, RhoB, and RhoC can be mono‐ADP‐ribosylated by C3 exoenzyme of Clostridium botulinum (C3bot). Quantitative profiling of ADP ribosylation of RhoA/B/C in HT22 cell lysates was achieved by incorporating SILAC‐labeled internal standard in LC–MS/MS analyses (Schroder et al, 2018). Besides, reactive oxygen species (ROS)‐ and reactive nitrogen species (RNS)‐mediated modifications of Cys118 in Ras proteins, for example, S ‐glutathionylation and S ‐nitrosylation, were recently profiled by LC–MS/MS analysis (Ntai et al, 2018; Messina, De Simone, & Ascenzi, 2019).…”

Section: Proteomic Studies Of Gtp‐binding Proteinsmentioning

confidence: 99%

Global and Targeted Profiling of Gtp‐binding Proteins in Biological Samples by Mass Spectrometry

Huang

Wang

2020

Mass Spectrometry Reviews

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GTP‐binding proteins are among the most important enzyme families that are involved in a plethora of biological processes. However, owing to the enormous diversity of the nucleotide‐binding protein family, comprehensive analyses of the expression level, structure, activity, and regulatory mechanisms of GTP‐binding proteins remain challenging with the use of conventional approaches. The many advances in mass spectrometry (MS) instrumentation and data acquisition methods, together with a variety of enrichment approaches in sample preparation, render MS a powerful tool for the comprehensive characterizations of the activities and expression levels of various GTP‐binding proteins. We review herein the recent developments in the application of MS‐based techniques, together with general and widely used affinity enrichment approaches, for the proteome‐wide and targeted capture, identification, and quantification of GTP‐binding proteins. The working principles, advantages, and limitations of various strategies for profiling the expression level, activity, posttranslational modifications, and interactome of GTP‐binding proteins are discussed. It can be envisaged that future applications of MS‐based proteomics will lead to a better understanding about the roles of GTP‐binding proteins in different biological processes and human diseases. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

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“…It is pivotal to identify SUMO‐modified substrates and SUMO acceptor sites at cell endogenous level for understanding SUMOylation‐involved biological processes. MS is a leading technology for investigating cellular proteomics, and PTMs . Over 200 types of PTMs have been reported, and at least 8 different modification forms have been exactly identified by MS, including acetylation, glycosylation, ubiquitylation, methylation, phosphorylation on serine and threonine (T), adenosine diphosphate ribosylation, and proline isomerization and so on .…”

Section: Ms Identification Of Sumo Modificationsmentioning

confidence: 99%

MS‐based strategies for identification of protein SUMOylation modification

Sheng

Wang

et al. 2019

Electrophoresis

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Protein SUMOylation modification conjugated with small ubiquitin-like modifiers (SUMOs) is one kind of PTMs, which exerts comprehensive roles in cellular functions, including gene expression regulation, DNA repair, intracellular transport, stress responses, and tumorigenesis. With the development of the peptide enrichment approaches and MS technology, more than 6000 SUMOylated proteins and about 40 000 SUMO acceptor sites have been identified. In this review, we summarize several popular approaches that have been developed for the identification of SUMOylated proteins in human cells, and further compare their technical advantages and disadvantages. And we also introduce identification approaches of target proteins which are co-modified by both SUMOylation and ubiquitylation. We highlight the emerging trends in the SUMOylation field as well. Especially, the advent of the clustered regularly interspaced short palindromic repeats/ Cas9 technique will facilitate the development of MS for SUMOylation identification.

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MS-based quantification of RhoA/B and RhoC ADP-ribosylation

Cited by 3 publications

References 17 publications

The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells

The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells

Global and Targeted Profiling of Gtp‐binding Proteins in Biological Samples by Mass Spectrometry

MS‐based strategies for identification of protein SUMOylation modification

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