scholarly journals Stoichiometric Thiol Redox Proteomics for Quantifying Cellular Responses to Perturbations

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 499
Author(s):  
Nicholas Day ◽  
Matthew Gaffrey ◽  
Wei-Jun Qian
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Aspect ◽  
Cysteine Residues ◽  
Protein Thiol ◽  
Absolute Quantitation ◽  
Post Translational Modifications ◽  
Redox Proteomics ◽  
Redox Biology ◽  
Thiol Redox ◽  
And Function

Post-translational modifications regulate the structure and function of proteins that can result in changes to the activity of different pathways. These include modifications altering the redox state of thiol groups on protein cysteine residues, which are sensitive to oxidative environments. While mass spectrometry has advanced the identification of protein thiol modifications and expanded our knowledge of redox-sensitive pathways, the quantitative aspect of this technique is critical for the field of redox proteomics. In this review, we describe how mass spectrometry-based redox proteomics has enabled researchers to accurately quantify the stoichiometry of reversible oxidative modifications on specific cysteine residues of proteins. We will describe advancements in the methodology that allow for the absolute quantitation of thiol modifications, as well as recent reports that have implemented this approach. We will also highlight the significance and application of such measurements and why they are informative for the field of redox biology.

scholarly journals RedoxiFluor: A microplate assay to quantify protein thiol redox state in percentages and moles

2021 ◽  
Author(s):  
Ahmet Tuncay ◽  
Anna Noble ◽  
Matthew Guille ◽  
James Cobley
Keyword(s):  
Redox State ◽  
Specific Protein ◽  
Microplate Assay ◽  
Protein Thiol ◽  
Redox Biology ◽  
Protein Thiols ◽  
Thiol Redox State ◽  
Thiol Redox ◽  
Cost Efficient ◽  
Context Specific

Abstract An accessible, time- and cost-efficient microplate assay to quantify protein thiol redox state in percentages and moles relative to the thiol proteome (i.e., context) and other targets (i.e., array mode) would be invaluable for understanding how protein thiols regulate essential biological processes. RedoxiFluor achieves several key benefits (i.e., percentages, moles, context, array mode) in a microplate format. After robustly validating RedoxiFluor, comparative analysis reveals that key benefits are intractable to other immunological techniques. Moles is an unprecedented achievement. Proof-of-concept studies illuminating fundamental redox principles (i.e., specificity, context, and heterogeneity) through measurement alone demonstrate how RedoxiFluor can advance understanding. For example, target specific protein thiol redox state changes are: (1) context specific (i.e., redox stimulus dependent); (2) selective (i.e., redox stimuli oxidise select targets); and (3) heterogenous (i.e., target responses vary markedly). RedoxiFluor is a powerful new tool for advancing a far-reaching and influential field: protein thiol redox biology.

Identification of Multiple Oxidative Post-Translational Modifications of Plasma Albumin in Patients with Pulmonary Hypertension of Sickle Cell Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4814-4814
Author(s):  
Adam Odhiambo ◽  
David Perlman ◽  
Martin Steinberg ◽  
Catherine E. Costello ◽  
Mark E. McComb ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Pulmonary Hypertension ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Plasma Proteins ◽  
Maldi Mass Spectrometry ◽  
Disease Pathogenesis ◽  
Post Translational Modifications ◽  
Redox Biology ◽  
Extensive Evaluation

Abstract Background: Pulmonary hypertension (PH) in sickle cell anemia (SCA) is characterized by decreased nitric oxide bioavailability; which may, in part, be related to increased oxidative stress. It is possible that via protein post-translational modifications, oxidants are able to affect both protein structure and function. We hypothesized that, in patients with SCA and PH, oxidative post-translational modifications (PTMs) occur on plasma proteins and are important in disease pathogenesis. We previously reported the identification of one PTM, a malondialdehyde adduct on peptide 146–159 of albumin and sought to do a more extensive evaluation of this protein to determine the presence of other abnormalities. Methods: Plasma was obtained from subjects with: SCA and PH (n=5); SCA steady-state without PH (n=4); Pulmonary Arterial Hypertension (PAH) (n=4); no evidence of cardiopulmonary disease (n=4). Platelet-poor plasma was separated into albumin-enriched and albumin-depleted fractions. The albumin-enriched fraction was subjected to proteolytic digestion by trypsin and studied by matrix-assisted-laser desorption/ionization (MALDI) mass spectrometry (MS) and liquid chromatography (LC)-MS/MS tandem mass spectrometry. Proteomic analyses were performed on all samples and post-translational modifications characterized by MS/MS. Results: We have characterized several additional peptides of albumin from patients with PH of SCA that bear lipid peroxidation and glycation adducts. Our comprehensive LC-MS/MS results have allowed us to identify 4-hydroxynonenal (HNE), hexose (HEX) and malonyl adduction at numerous, distinct cysteine, histidine, lysine, and threonine albumin residues. Conclusion: Increased oxidant burden and altered redox biology is characteristic of PH in SCA. Plasma proteins, such as albumin, are a target for oxidants and changes in their structure may play a role in disease pathogenesis.

PROTEIN DYNAMICS IN PHOSPHORYLATION-MEDIATED ALLOSTERY PROBED BY AMIDE EXCHANGE MASS SPECTROMETRY

COSMOS ◽  
2013 ◽  
Vol 09 (01) ◽  
pp. 19-27
Author(s):  
MADHUBRATA GHOSH ◽  
GANESH S. ANAND
Keyword(s):  
Mass Spectrometry ◽  
Protein Dynamics ◽  
Protein Function ◽  
Post Translational Modifications ◽  
Exchange Mass Spectrometry ◽  
Amide Exchange ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
And Function

A major goal of molecular biology is to correlate molecular structure with function. Since most enzymes and biological catalysts are proteins, the focus for correlating 'form' with 'function' has been entirely on protein macromolecular structure. It is obvious that any understanding of protein function must come through an understanding protein dynamics. Furthermore, all of the regulatory reactions are through changes in dynamics brought about by post-translational modifications, the most important of which is phosphorylation. This review highlights the important role of covalent phosphorylation and noncovalent phosphates in regulating allosteric effects and function through a study of protein dynamics. Mass spectrometry is a relatively new and increasingly important tool for describing protein dynamics. All examples described in this review have been studied by amide hydrogen/deuterium exchange mass spectrometry.

scholarly journals Discovery of lysine post-translational modifications through mass spectrometric detection

2012 ◽  
Vol 52 ◽  
pp. 147-163 ◽  
Author(s):  
Barry M. Zee ◽  
Benjamin A. Garcia
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Mass Spectrometric ◽  
Mass Spectrometric Detection ◽  
Post Translational Modifications ◽  
Target Set ◽  
Modified Proteins ◽  
And Function

The complexity of an organism's proteome is in part due to the diversity of post-translational modifications present that can direct the location and function of a protein. To address the growing interest in characterizing these modifications, mass spectrometric-based proteomics has emerged as one of the most essential experimental platforms for their discovery. In searching for post-translational modifications within a target set of proteins to global surveys of particularly modified proteins within a given proteome, various experimental MS (mass spectrometry) and allied techniques have been developed. Out of 20 naturally encoded amino acids, lysine is essentially the most highly post-translationally modified residue. This chapter provides a succinct overview of such methods for the characterization of protein lysine modifications as broadly classified, such as methylation and ubiquitination.

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.

Unrestrictive protein modification localization and quality control for open search of mass spectra

2018 ◽  
Author(s):  
Zhiwu An ◽  
Fuzhou Gong ◽  
Yan Fu
Keyword(s):  
Mass Spectrometry ◽  
Quality Control ◽  
Tandem Mass Spectrometry ◽  
Mass Spectra ◽  
Protein Modification ◽  
Software Tool ◽  
Tandem Mass ◽  
Simulation Data ◽  
Post Translational Modifications

We have developed PTMiner, a first software tool for automated, confident filtering, localization and annotation of protein post-translational modifications identified by open (mass-tolerant) search of large tandem mass spectrometry datasets. The performance of the software was validated on carefully designed simulation data. <br>

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