scholarly journals Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes – a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC-MS

2021 ◽  
Author(s):  
Alienke van Pijkeren ◽  
Jörn Dietze ◽  
Alejandro Sánchez Brotons ◽  
Tim Lijster ◽  
Andrei Barcaru ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Protein Modification ◽  
High Resolution Mass Spectrometry ◽  
Reaction Rates ◽  
Lysine Acetylation ◽  
Chemical Labeling ◽  
Comprehensive Description ◽  
Site Specific ◽  
New Approach

Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated enabling accurate relative quantification of site-specific lysine acetylation in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics. <br>

scholarly journals Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes – a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC-MS

2021 ◽  
Author(s):  
Alienke van Pijkeren ◽  
Jörn Dietze ◽  
Alejandro Sánchez Brotons ◽  
Tim Lijster ◽  
Andrei Barcaru ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Protein Modification ◽  
High Resolution Mass Spectrometry ◽  
Reaction Rates ◽  
Lysine Acetylation ◽  
Chemical Labeling ◽  
Comprehensive Description ◽  
Site Specific ◽  
New Approach

Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated enabling accurate relative quantification of site-specific lysine acetylation in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics. <br>

scholarly journals Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes – a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC-MS

2021 ◽  
Author(s):  
Alienke van Pijkeren ◽  
Jörn Dietze ◽  
Alejandro Sánchez Brotons ◽  
Tim Lijster ◽  
Andrei Barcaru ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Protein Modification ◽  
High Resolution Mass Spectrometry ◽  
Reaction Rates ◽  
Lysine Acetylation ◽  
Chemical Labeling ◽  
Comprehensive Description ◽  
Site Specific ◽  
New Approach

Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated enabling accurate relative quantification of site-specific lysine acetylation in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics. <br>

scholarly journals Natural isotope correction improves analysis of protein modification dynamics

2021 ◽  
Author(s):  
Jörn Dietze ◽  
Alienke van Pijkeren ◽  
Anna-Sophia Egger ◽  
Mathias Ziegler ◽  
Marcel Kwiatkowski ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Modification ◽  
High Resolution Mass Spectrometry ◽  
Accurate Determination ◽  
Mass Spectrometry Data ◽  
Stable Isotope Labelling ◽  
Isotope Labelling ◽  
Naturally Occurring ◽  
Correct Estimation

AbstractStable isotope labelling in combination with high-resolution mass spectrometry approaches are increasingly used to analyze both metabolite and protein modification dynamics. To enable correct estimation of the resulting dynamics, it is critical to correct the measured values for naturally occurring stable isotopes, a process commonly called isotopologue correction or deconvolution. While the importance of isotopologue correction is well recognized in metabolomics, it has received far less attention in proteomics approaches. Although several tools exist that enable isotopologue correction of mass spectrometry data, the majority is tailored for the analysis of low molecular weight metabolites. We here present PICor which has been developed for isotopologue correction of complex isotope labelling experiments in proteomics or metabolomics and demonstrate the importance of appropriate correction for accurate determination of protein modifications dynamics, using histone acetylation as an example.

scholarly journals The interaction of canonical Wnt/β-catenin signaling with protein lysine acetylation

2022 ◽  
Vol 27 (1) ◽  
Author(s):  
Hongjuan You ◽  
Qi Li ◽  
Delong Kong ◽  
Xiangye Liu ◽  
Fanyun Kong ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Protein Function ◽  
Protein Modification ◽  
Epigenetic Modification ◽  
Lysine Acetylation ◽  
Protein Acetylation ◽  
Post Translational Modification ◽  
Histone Protein ◽  
Protein Lysine Acetylation ◽  
Canonical Wnt

AbstractCanonical Wnt/β-catenin signaling is a complex cell-communication mechanism that has a central role in the progression of various cancers. The cellular factors that participate in the regulation of this signaling are still not fully elucidated. Lysine acetylation is a significant protein modification which facilitates reversible regulation of the target protein function dependent on the activity of lysine acetyltransferases (KATs) and the catalytic function of lysine deacetylases (KDACs). Protein lysine acetylation has been classified into histone acetylation and non-histone protein acetylation. Histone acetylation is a kind of epigenetic modification, and it can modulate the transcription of important biological molecules in Wnt/β-catenin signaling. Additionally, as a type of post-translational modification, non-histone acetylation directly alters the function of the core molecules in Wnt/β-catenin signaling. Conversely, this signaling can regulate the expression and function of target molecules based on histone or non-histone protein acetylation. To date, various inhibitors targeting KATs and KDACs have been discovered, and some of these inhibitors exert their anti-tumor activity via blocking Wnt/β-catenin signaling. Here, we discuss the available evidence in understanding the complicated interaction of protein lysine acetylation with Wnt/β-catenin signaling, and lysine acetylation as a new target for cancer therapy via controlling this signaling.

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.

The Dudley Lee Case: A new approach to factual causation and its implications for transformative jurisprudence

2017 ◽  
Vol 30 (1) ◽  
pp. 273-289
Author(s):  
Anmari Meerkotter
Keyword(s):  
Case Note ◽  
Constitutional Court ◽  
Recent Contribution ◽  
New Approach ◽  
State Accountability ◽  
The Law ◽  
Transformative Impact ◽  
Constitutional Jurisprudence ◽  
Law Of Delict

The Constitutional Court (CC) judgment of Lee v Minister of Correction Services 2013 2SA 144 (CC) is a recent contribution to transformative constitutional jurisprudence in the field of the law of delict. This matter turned on the issue of factual causation in the context of wrongful and negligent systemic omissions by the state. In this case note, I explore the law relating to this element of delictual liability with specific regard to the traditional test for factual causation – the conditio sine qua non (‘but-for’) test. In particular, I note the problems occasioned by formalistic adherence to this test in the context of systemic state omissions as evidenced by the SCA judgment in the same matter. I also consider the manner in which English courts have addressed this problem. Thereafter, I analyse the CC’s broader approach to the determination of factual causation as one based on common sense and justice. I argue that this approach endorses a break from a formalistic application of the test and constitutes a step towards an approach which resonates with the foundational constitutional values of freedom, dignity and equality. Furthermore, it presents an appropriate solution to the problems associated with factual causation where systemic omissions are concerned. I then consider the transformative impact of the Lee judgment. In particular, I argue that the broader enquiry favoured by the CC facilitates the realisation of constitutionally guaranteed state accountability, and amounts to an extension of the existing norm of accountability jurisprudence. Hence, I contend that the judgment presents a further effort by the Constitutional Court to effect wholesale the constitutionalisation of the law of delict, as well as a vindicatory tool to be used by litigants who have been adversely affected by systemic state omissions.

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

2018 ◽  
Author(s):  
Daniel D. Brauer ◽  
Emily C. Hartman ◽  
Daniel L.V. Bader ◽  
Zoe N. Merz ◽  
Danielle Tullman-Ercek ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Modification ◽  
Positive Charge ◽  
Specific Protein ◽  
High Yield ◽  
Site Specific ◽  
Protein Cage ◽  
Protein Carriers ◽  
Site Selective ◽  
Targeted Library

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

Determination of quaternary ammonium compounds in food products by the method of ultra-performance liquid chromatography/high resolution mass-spectrometry

2020 ◽  
Vol 86 (8) ◽  
pp. 23-31
Author(s):  
V. G. Amelin ◽  
D. S. Bolshakov
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Quaternary Ammonium ◽  
High Resolution Mass Spectrometry ◽  
Food Products ◽  
Quaternary Ammonium Compounds ◽  
High Resolution Mass ◽  
Ammonium Compounds ◽  
Resolution Mass

The goal of the study is developing a methodology for determination of the residual amounts of quaternary ammonium compounds (QAC) in food products by UHPLC/high-resolution mass spectrometry after water-acetonitrile extraction of the determined components from the analyzed samples. The identification and determination of QAC was carried out on an «UltiMate 3000» ultra-high-performance liquid chromatograph (Thermo Scientific, USA) equipped with a «maXis 4G» high-resolution quadrupole-time-of-flight mass spectrometric detector and an ion spray «ionBooster» source (Bruker Daltonics, Germany). Samples of milk, cheese (upper cortical layer), dumplings, pork, chicken skin and ground beef were used as working samples. Optimal conditions are specified for chromatographic separation of the mixture of five QAC, two of them being a mixture of homologues with a linear structure (including isomeric forms). The identification of QAC is carried out by the retention time, exact mass of the ions, and coincidence of the mSigma isotopic distribution. The limits for QAC detection are 0.1 – 0.5 ng/ml, the determination limits are 1 ng/ml for aqueous standard solutions. The determinable content of QAC in food products ranges within 1 – 100 ng/g. The results of analysis revealed the residual amount of QAC present in all samples, which confirms data of numerous sources of information about active use of QAC-based disinfectants in the meat and dairy industry. The correctness of the obtained results is verified by introduction of the additives in food products at a level of 10 ng/g for each QAC. The relative standard deviation of the analysis results does not exceed 0.18. The duration of the analysis is 30 – 40 min.

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