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Cited by 348Redox biology and oxidative stressAntioxidant Activity and Oxidative StressAdvanced Glycation End Products research

Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases

Open access
Kehm, Richard, Baldensperger, Tim, Raupbach, Jana, Höhn, Annika·2021·Redox Biology
DOI 10.1016/j.redox.2021.101901PMC PMC8113053
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Abstract

Generation of reactive oxygen species and related oxidants is an inevitable consequence of life. Proteins are major targets for oxidation reactions, because of their rapid reaction rates with oxidants and their high abundance in cells, extracellular tissues, and body fluids. Additionally, oxidative stress is able to degrade lipids and carbohydrates to highly reactive intermediates, which eventually attack proteins at various functional sites. Consequently, a wide variety of distinct posttranslational protein modifications is formed by protein oxidation, glycoxidation, and lipoxidation. Reversible modifications are relevant in physiological processes and constitute signaling mechanisms ("redox signaling"), while non-reversible modifications may contribute to pathological situations and several diseases. A rising number of publications provide evidence for their involvement in the onset and progression of diseases as well as aging processes. Certain protein oxidation products are chemically stable and formed in large quantity, which makes them promising candidates to become biomarkers of oxidative damage. Moreover, progress in the development of detection and quantification methods facilitates analysis time and effort and contributes to their future applicability in clinical routine. The present review outlines the most important classes and selected examples of oxidative protein modifications, elucidates the chemistry beyond their formation and discusses available methods for detection and analysis. Furthermore, the relevance and potential of protein modifications as biomarkers in the context of disease and aging is summarized.

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Written from the paper’s full text.

TL;DR

This review summarizes the chemistry, detection methods, and biomarker potential of oxidative protein modifications, including sulfur/aromatic amino acid oxidation, glycoxidation, lipoxidation, carbonylation, and nitration. It concludes that protein carbonyls and 3-nitrotyrosine are promising biomarkers, while many routinely used assays remain insufficiently specific or standardized for clinical use.

Key findings

Proteins are estimated to scavenge 50%–75% of generated reactive species, making them major oxidation targets. The review describes reversible modifications such as cysteine disulfides and methionine sulfoxide as relevant to redox signaling, and irreversible products such as cysteine sulfonic acid, dityrosine, CML, pentosidine, 4-HNE/MDA adducts, protein carbonyls, and 3-nitrotyrosine as disease/aging biomarker candidates. Aminoadipic and glutamic semialdehyde are reported to account for about 60% of total protein carbonylation in liver. Clinical studies cited mostly show elevated protein carbonyls and 3-nitrotyrosine in multiple diseases, but results for MDA, AOPP, and some glycoxidation products are inconsistent; the authors conclude MDA is not a suitable marker for human aging and age-related diseases.

Methods

Narrative review of protein oxidation mechanisms, detection/quantification methods, and biomarker relevance in human disease and aging. For the clinical biomarker section, the authors searched PubMed in November 2020 using individual biomarker terms plus “human disease,” “human age-related disease,” or “human aging,” and focused on recent clinical studies from 2015–2020. The review summarizes selected studies in tables by biomarker class, specimen, study population, method, and main outcome.

Limitations

The paper states that many frequently used routine methods are “less than optimal,” that the clinical relevance of most protein modifications as biomarkers is questionable, and that further longitudinal studies with standardized methods are needed. It also states that immunoassays often lack full chemical/structural characterization and that AOPP spectrophotometric assays are highly unspecific. Evident from the text: this is a narrative review, not a systematic review or meta-analysis, and the cited clinical studies are heterogeneous in methods, specimens, and sample sizes, including several very small groups.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.redox.2021.101901

Cites

249 of 249 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

112 of 216 cited sources checked · 53 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Sulfur-containing moieties such as the sulfhydryl group of cysteine and the thioether group of methionine are targeted by oxidative stress and form a plethora of posttranslational protein modifications.

    “Sulfur-containing moieties such as the sulfhydryl group of cysteine and the thioether group of methionine are targeted by oxidative stress and a plethora of posttranslational protein modifications is formed (Fig. 1) [15].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [15] Ryan, 2014, Oxidative post-translational modifications and their involvement in the pathogenesis of autoimmune diseases, Redox Biol. source
  • Premisecitation

    A higher level of oxidative damage may arise from increased oxidant generation and/or a decrease or failure of defense systems under conditions such as diseases and aging.

    “Furthermore, a higher level of damage may arise from increased oxidant generation and/or a decrease or failure of defense systems under several conditions such as diseases and aging [3,4].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [3] Kregel, 2007, An integrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations, Am. J. Physiol. Regul. Integr. Comp. Physiol. source
    • cites [4] Liguori, 2018, Oxidative stress, aging, and diseases, Clin. Interv. Aging source
  • Premisecitation

    The main product of tyrosyl radical reaction after enolization is the fluorescent protein crosslink ortho,ortho-dityrosine.

    “After enolization, the main product of this reaction is the fluorescent protein crosslink ortho,ortho-dityrosine [23].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [23] Heinecke, 1993, Tyrosyl radical generated by myeloperoxidase catalyzes the oxidative cross-linking of proteins, J. Clin. Invest. source

      The Introduction (page not provided) calls o,o′-dityrosine the major product of the tyrosyl-radical reaction, and the Methods describe protein-bound dityrosine measured by fluorescence. Our copy does not describe enolization as a step preceding its formation. (LLM, conf=0.88)

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  • Premisecitation

    The irreversible modification 3-nitrotyrosine is formed by nitration of tyrosine via attack of the nitrogen dioxide radical at the ortho-position of the aromatic ring.

    “The irreversible modification 3-nitrotyrosine (3-NT) is formed by nitration of tyrosine via attack of the nitrogen dioxide radical at the ortho-position of the aromatic ring [74].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [74] Bartesaghi, 2018, Fundamentals on the biochemistry of peroxynitrite and protein tyrosine nitration, Redox Biol. source

      The “Nitration as a free radical process” section (page unavailable) states that nitration places a nitro group at position 3 of tyrosine’s phenolic ring and that the tyrosine radical reacts with •NO2 to yield 3-nitrotyrosine. It does not establish that the resulting 3-nitrotyrosine modification is irreversible; its discussion of reversal concerns the tyrosine-radical intermediate, not the nitrated product. (LLM, conf=0.85)

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  • Premisecitation

    Glyoxal is mainly formed by oxidative degradation of carbohydrates, lipids, nucleotides, and serine.

    “Although the latter mechanism is non-oxidative, the reactive precursor glyoxal is mainly formed by oxidative degradation of carbohydrates, lipids, nucleotides, and serine [37].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [37] Rabbani, 2015, Dicarbonyl stress in cell and tissue dysfunction contributing to ageing and disease, Biochem. Biophys. Res. Commun. source

      Table 1 (appearing in our copy under Closing remarks) lists lipid peroxidation, oxidative degradation of serine, monosaccharide degradation, and nucleotide degradation as sources of glyoxal. It does not say that all four pathways are oxidative or that they are collectively the main sources; it also lists degradation of glycated proteins and food and beverages. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • May combine two claims. 60
  • Classified as probably not a substantial claim. 25
  • The quote only partly states this claim. 2

  • May combine two claims.

    Premisecitation

    The overoxidation product cysteine sulfonic acid is irreparably damaged and the affected protein has to be degraded by the proteasome.

    “The overoxidation product cysteine sulfonic acid is irreparably damaged and the affected protein has to be degraded by the proteasome [19].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [19] Xiong, 2011, S-glutathionylation: from molecular mechanisms to health outcomes, Antioxidants Redox Signal. source

      Section III, “Enzymes That Catalyze the S-Glutathionylation Cycle” (page not available), says oxidation to sulfonic acid is generally irreparable and leads to proteasomal degradation of the protein. The citing sentence omits “generally” and makes degradation categorical (“has to”), overstating the review’s qualification. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Tyrosine is redox active; its phenolic side-chain is easily oxidized because the intermediary tyrosyl radical is stabilized by mesomeric delocalization.

    “In particular, tyrosine is a redox active structure. The phenolic side-chain is easily oxidized, because the intermediary tyrosyl radical is stabilized by mesomeric delocalization of the unpaired electron (Fig. 2A) [22].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [22] Correia, 2012, UV-light exposure of insulin: pharmaceutical implications upon covalent insulin dityrosine dimerization and disulphide bond photolysis, PLoS One source

      The Introduction (page unavailable) describes electron loss from excited tyrosine followed by formation of a tyrosyl radical, and the Discussion (page unavailable) calls that radical relatively stable. These passages support tyrosine's redox activity, but do not say that its phenolic side-chain is easily oxidized or attribute radical stability to mesomeric delocalization. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Reaction of cysteine with another cysteine under non-enzymatic and enzyme-mediated conditions leads to cystine, which stabilizes protein structures via intra- and intermolecular disulfide bridges.

    “Reaction of cysteine with another cysteine under non-enzymatic as well as enzyme mediated conditions leads to cystine. Cystine is a vitally important modification, because it stabilizes protein structures via intra- and intermolecular disulfide bridges.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [17] Tu, 2004, Oxidative protein folding in eukaryotes mechanisms and consequences, JCB (J. Cell Biol.) source

      The Abstract (page not provided) says disulfide bonds involving cysteine residues are often crucial to protein stability and describes both spontaneous formation in vitro and assisted formation in vivo. The section on oxidative folding machinery also describes enzyme-catalyzed disulfide formation. Our copy does not explicitly identify the product as cystine or establish the claim’s distinction between intra- and intermolecular bridges. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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    Premisecitation

    Highly reactive species damage numerous sites at side-chains and backbones of proteins, while less reactive species have higher selectivity regarding targeted residues.

    “Highly reactive species damage numerous sites at side-chains and backbones of proteins, while less reactive species have higher selectivity regarding targeted residues [7].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [7] Davies, 2016, Protein oxidation and peroxidation, Biochem. J. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Nitric oxide is an important signaling molecule involved in vasodilation and neurotransmission via activation of soluble guanylate cyclase and generation of the second messenger cGMP.

    “Nitric oxide (•NO) is an important signaling molecule involved in vasodilation and neurotransmission via activation of soluble guanylate cyclase and generation of the second messenger cGMP [69].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [69] Murad, 1986, Cyclic guanosine monophosphate as a mediator of vasodilation, J. Clin. Invest. source

      The paper’s Figure 1 caption states that nitric oxide derived from nitrovasodilators activates soluble guanylate cyclase, increases cyclic GMP synthesis, and leads to vascular relaxation. The passages in our copy do not address nitric oxide’s role in neurotransmission, so that part of the claim is unsupported. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Unbound DNPH absorbs at the same wavelength as hydrazones (λ = 370 nm), requiring washing steps that cause an inevitable loss of protein of up to 10-15%.

    “Unbound DNPH needs to be removed from the sample, because it absorbs at the same wavelength as hydrazones (λ = 370 nm). Several washing steps of the protein are required to remove unbound DNPH leading to an inevitable loss of protein of up to 10–15% [75].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [75] Rogowska-Wrzesinska, 2014, Analysis of protein carbonylation--pitfalls and promise in commonly used methods, Free Radic. Res. source

      The indexed passages are bibliographic records and keywords, with no substantive passage on DNPH absorbance or washing-related protein loss. Searches for DNPH, 370 nm, and 10–15% protein loss found no passage that verifies the claim. (LLM, conf=0.72)

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ALEs are protein modifications formed by nucleophilic reaction of proteins with reactive carbonyl species originating from oxidative lipid degradation.

    “ALEs are protein modifications formed by the nucleophilic reaction of proteins with reactive carbonyl species (RCS) originating from oxidative lipid degradation [49].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [49] Gianazza, 2019, Lipoxidation in cardiovascular diseases, Redox Biol. source

  • May combine two claims.

    Premisecitation

    4-Hydroxy-2-nonenal (4-HNE) forms stable Michael adducts with cysteine and histidine; nucleophilic attack of lysine forms unstable Michael adducts that require reduction prior to analysis.

    “This positive partial charge is attacked by nucleophiles such as cysteine and histidine and stable Michael adducts are formed (Fig. 4B) [50]. Nucleophilic attack of lysine forms unstable Michael adducts, which require reduction, e.g., by sodium borohydride prior to analysis [57].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [50] Vistoli, 2013, Advanced glycoxidation and lipoxidation end products (AGEs and ALEs): an overview of their mechanisms of formation, Free Radic. Res. source
    • cites [57] Requena, 1997, Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein, Biochem. J. source

  • May combine two claims.

    Premisecitation

    Top-down MS can evaluate the total modification status on an individual protein, but has disadvantages including low sequence coverage in biological samples, incompatibility with detergents, limitation to individual known proteins, and high protein amount requirements.

    “Top-down approaches can be helpful to evaluate the total modification status on an individual protein [[93], [94], [95], [96]].”
    • Also stated in: § Analysis of specific oxidation products · plain
      “For example, sequence coverage in biological samples remains quite low using top-down MS due to overlapping and deconvolution effects [79].”
    • Also stated in: § Analysis of specific oxidation products · plain
      “Finally, top-down analysis requires high amounts of purified proteins to acquire highly resolved peaks of precursor and product ions with sufficient intensity [97].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [79] Verrastro, 2015, Mass spectrometry-based methods for identifying oxidized proteins in disease: advances and challenges, Biomolecules source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [97] Siuti, 2007, Decoding protein modifications using top-down mass spectrometry, Nat. Methods source

  • May combine two claims.

    Premisecitation

    LC-MS/MS has been used for proteomic analysis to identify carbonylated proteins, providing a more sensitive approach, but reveals limitations for quantitative assessments.

    “Besides this, LC-MS/MS has been also used for proteomic analysis to identify carbonylated proteins, providing a more sensitive approach, but reveal limitations for quantitative assessments [86].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [86] Augustyniak, 2015, Validation of protein carbonyl measurement: a multi-centre study, Redox Biol source

      The Results and discussion section reports using LC–MS/MS to identify carbonylated proteins and says the method used provided no quantitative information. The Conclusions likewise describe poorly developed quantitative applications for MS. The paper does not establish the claim that LC–MS/MS is a more sensitive approach. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Aminoadipic and glutamic semialdehyde are examples of direct oxidation of amino acids and are responsible for about 60% of total protein carbonylation in liver.

    “Aminoadipic and glutamic semialdehyde are examples for direct oxidation of amino acids and responsible for about 60% of total protein carbonylation in liver [64].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [64] Requena, 2001, Glutamic and aminoadipic semialdehydes are the main carbonyl products of metal-catalyzed oxidation of proteins, Proc. Natl. Acad. Sci. Unit. States Am. source

  • May combine two claims.

    Premisecitation

    Several methods for detection of glycoxidation products in blood, urine, and tissue samples have been established, including spectrophotometry, IHC, immunoblot, and ELISA, with ELISA most commonly used.

    “Several methods for their detection in blood, urine, and tissue samples have been established in recent years, such as spectrophotometry, immunohistochemistry (IHC), immunoblot, and ELISA techniques, whereas the latter has been most commonly used.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [158] Ashraf, 2015, Recent advances in detection of AGEs: immunochemical, bioanalytical and biochemical approaches, IUBMB Life source

  • May combine two claims.

    Premisecitation

    Cysteine residues are major targets of ROS and undergo many posttranslational modifications, leading to functional and structural alterations of proteins.

    “In particular, cysteine residues are major targets of ROS undergoing a large number of posttranslational modifications, leading to functional and structural alterations of proteins.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [118] Bin, 2017, Oxidation resistance of the sulfur amino acids: methionine and cysteine, BioMed Res. Int. source

      Section 3 (Cysteine; page unavailable) says cysteine residues are easily oxidized and react with H2O2, supporting their susceptibility to ROS. It does not establish that they are major ROS targets or describe numerous posttranslational modifications leading to functional and structural changes in proteins. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [119] Brosnan, 2006, The sulfur-containing amino acids: an overview, J. Nutr. source

      The discussion of cysteine in proteins (p. 1636S) says cysteine residues form disulfide bonds that are important to protein structure and folding. It does not say cysteine residues are major ROS targets or undergo a large number of ROS-related posttranslational modifications; its explicit discussion of ROS-related residue oxidation concerns methionine instead. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Guerin-Duborg et al. used an AGE-ELISA Kit preferentially quantifying CML and found no differences between non-diabetic subjects and T2D patients, or between T2D patients with and without vascular diseases.

    “Guerin-Duborg and colleagues used an AGE-ELISA Kit preferentially quantifying CML and revealed no differences between non-diabetic subjects and T2D patients as well as T2D patients with and without vascular diseases.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [160] Guerin-Dubourg, 2017, Association between fluorescent advanced glycation end-products and vascular complications in type 2 diabetic patients, BioMed Res. Int. source

  • May combine two claims.

    Premisecitation

    Skin AF has been applied in clinical settings, indicating elevated AGE levels in several diseases and aging.

    “has been applied in clinical settings, indicating elevated AGE levels in several diseases and aging, as shown in Table 5”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [161] Stirban, 2014, Skin autofluorescence - a non-invasive measurement for assessing cardiovascular risk and risk of diabetes, Eur. Endocrinol. source

  • May combine two claims.

    Premisecitation

    AGEs, including glycoxidation products, play a fundamental role in human aging and in aging of vertebrate and invertebrate model organisms.

    “The fundamental role of AGEs, including glycoxidation products, in human aging, but also in aging of vertebrate and invertebrate model organism has been reviewed in 2018 by Chaudhuri and colleagues [153]”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [153] Chaudhuri, 2018, The role of advanced glycation end products in aging and metabolic diseases: bridging association and causality, Cell Metabol. source

      The Abstract describes AGE accumulation as an inevitable component of aging in humans and other eukaryotes, and the model-organism section reviews vertebrate and invertebrate studies. But the Abstract says AGEs are linked to, and only perhaps responsible for, aging-related changes; the Conclusions say their causal effects remain poorly understood. Calling their role in aging “fundamental” states that role more firmly than the review does. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lipid peroxidation has been implicated in a high number of human pathologic conditions.

    “The biological process of lipid peroxidation has been implicated in a high number of human pathologic conditions [172,173].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [172] Tsikas, 2017, Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: analytical and biological challenges, Anal. Biochem. source
    • cites [173] Ramana, 2019, Lipid peroxidation products in human health and disease 2019, Oxid Med Cell Longev source

  • May combine two claims.

    Premisecitation

    O'Grady et al. found higher CML levels in plasma of T2D patients compared to healthy subjects but similar pentosidine concentrations.

    “O'Grady et al. found higher CML levels in plasma of T2D patients compared to healthy subjects but similar pentosidine concentrations [165].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [165] O'Grady, 2020, Development and application of mass spectroscopy assays for nepsilon-(1-carboxymethyl)-L-lysine and pentosidine in renal failure and diabetes, J Appl Lab Med source

      The “Control versus T2DM patients” results (page unavailable) report significantly higher CML in T2DM patients and no significant difference in pentosidine. However, the paper identifies the measured samples as serum, not plasma, so it does not establish the claim specifically for plasma. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Antibodies against MDA-modified proteins are commercially available and highly selective MS methods for their detection have been established.

    “To our knowledge, MDA modified proteins have not been used as biomarkers in recent clinical investigations although antibodies are commercially available and highly selective MS methods have been established, previously [57].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [57] Requena, 1997, Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein, Biochem. J. source

      The Introduction (page not provided) says antibodies are available for measuring MDA epitopes and describes a sensitive, specific GC/MS assay for MDA–lysine adducts in proteins. It does not say the antibodies are commercially available. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Baseline parameters and methods used in the Ravassa and Segre studies were similar.

    “For instance, while Ravassa et al. found higher 3-NT levels in T2D patients with additional cardiovascular events, no differences were observed by Segre and colleagues although baseline parameters of the study populations and the used methods were similar [189,190].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [189] Ravassa, 2015, Association of low GLP-1 with oxidative stress is related to cardiac disease and outcome in patients with type 2 diabetes mellitus: a pilot study, Free Radic. Biol. Med. source
    • cites [190] Segre, 2015, Troponin in diabetic patients with and without chronic coronary artery disease, BMC Cardiovasc. Disord. source

      The Results report no significant difference in nitrotyrosine between Segre’s diabetic patients with coronary artery disease and controls (27.9 versus 28.7; p = 0.77). The Methods and Results describe Segre’s participants and assay, but our copy does not compare their baseline characteristics or methods with Ravassa’s study, so the claimed similarity is not established by this source. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • The quote only partly states this claim.

    Premisecitation

    In patients with interstitial lung disease, 3-NT levels are elevated compared to healthy individuals (plasma, MS/MS).

    “Elevated levels of 3-NT in patients with interstitial lung disease”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [142] Pennathur, 2016, Oxidative modifications of protein tyrosyl residues are increased in plasma of human subjects with interstitial lung disease, Am. J. Respir. Crit. Care Med. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Proteins can scavenge a majority (50%-75%) of generated reactive species.

    “It has been estimated that proteins can scavenge a majority (50%–75%) of generated reactive species [6].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6] Davies, 1999, Stable markers of oxidant damage to proteins and their application in the study of human disease, Free Radic. Biol. Med. source

  • May combine two claims.

    Premisecitation

    The variety of reaction sites generates a wide range of posttranslational protein modifications, changing composition and folding, net charge, and hydrophobicity/hydrophilicity, and affecting protein functions as receptors, enzymes, carrier or structural proteins.

    “The variety of reaction sites generates a wide range of posttranslational protein modifications consequently changing composition and folding, the net charge, as well as the hydrophobicity/hydrophilicity of proteins. This affects their functions as receptors, enzymes, carrier or structural proteins [8].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] Droge, 2002, Free radicals in the physiological control of cell function, Physiol. Rev. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    Under normal circumstances, formation and subsequent degradation of reactive species is regulated by cellular defense systems including scavenging enzymes and non-enzymatic antioxidants.

    “Under normal circumstances, the formation and subsequent degradation of reactive species is regulated by cellular defense systems, including scavenging enzymes able to remove oxidants or their precursors such as superoxide dismutases, catalase, thioredoxin/thioredoxin reductase, and glutathione peroxidase.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Reversible protein modifications are relevant in physiological processes and constitute signaling mechanisms (redox signaling), while non-reversible modifications may contribute to pathological situations and several diseases.

    “Reversible modifications are relevant in physiological processes and constitute signaling mechanisms (“redox signaling”), while non-reversible modifications may contribute to pathological situations and several diseases.”
    • Also stated in: § Introduction · boosted
      “It has been established that reversible modifications are thought to be relevant in physiological processes and constitute signaling mechanisms under appropriate conditions (“redox signaling”), while non-reversible modifications may contribute to pathological situations and several diseases [9].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [9] Spickett, 2012, Protein oxidation: role in signalling and detection by mass spectrometry, Amino Acids source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    Protein oxidation products can be determined in blood and urine samples, but also in specific tissue or cell samples.

    “Protein oxidation products can be determined in blood and urine samples, but also in specific tissue or cell samples.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Sample availability is an important factor limiting the reliability of a biomarker.

  • May combine two claims.

    Premisecitation

    Nitric oxide reacts with superoxide at a high rate constant to give peroxynitrite, which can decompose spontaneously to yield nitrogen dioxide and hydroxyl radicals.

    “However, nitric oxide reacts with superoxide at a high rate constant to give peroxynitrite (ONOO−), able to decompose spontaneously to yield nitrogen dioxide (•NO2) and hydroxyl radicals [2].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Hughes, 1970, A possible role for the species peroxonitrite in nitrification, Biochim. Biophys. Acta source

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationreasoning

    Progress in detection and quantification methods facilitates analysis time and effort and contributes to future applicability of protein oxidation products in clinical routine.

    “Moreover, progress in the development of detection and quantification methods facilitates analysis time and effort and contributes to their future applicability in clinical routine.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: To function as suitable biomarkers, protein oxidation products must be stable, accumulate in detectable concentrations, and correlate with disease severity.

  • May combine two claims.

    Premisecitation

    Formation of cysteine thiyl radical and sulfenic acid is a reversible process; both intermediates are highly unstable and serve as precursors for several oxidized cysteine modifications.

    “Formation of cysteine thiyl radical and sulfenic acid is a reversible process and both intermediates are highly unstable. Both serve as precursors for several oxidized cysteine modifications (Fig. 1A) [16].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Trujillo, 2016, One-and two-electron oxidation of thiols: mechanisms, kinetics and biological fates, Free Radic. Res. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Hydroxyl radical is formed from superoxide and hydrogen peroxide in the presence of metal ions (Fenton reaction) and has a very short half-life of approximately 10−9 s.

    “Additionally, important primary oxygen-containing compounds with reactive properties are superoxide (O2•-) and the most reactive hydroxyl radical (•OH), formed from O2•- and hydrogen peroxide (H2O2) in the presence of metal ions (Fenton reaction) with a very short half-life of approximately 10−9 s [1].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Sies, 1993, Strategies of antioxidant defense, Eur. J. Biochem. source

  • May combine two claims.

    Premisecitation

    Methionine is reversibly oxidized to methionine sulfoxide and irreversibly oxidized to methionine sulfone; methionine sulfone is stable and not targeted by methionine sulfoxide reductases.

    “In contrast to the non-enzymatic oxidation of methionine to methionine sulfoxide, the reduction is catalyzed by methionine sulfoxide reductases [20]. Further oxidation results in methionine sulfone formation, which is not targeted by methionine sulfoxide reductases and has to be considered as a stable modification [21].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [20] Vogt, 1995, Oxidation of methionyl residues in proteins: tools, targets, and reversal, Free Radic. Biol. Med. source
    • cites [21] Ahmed, 2003 source

  • May combine two claims.

    Premisecitation

    Protein carbonylation is a stable modification induced by ROS via three pathways: direct oxidation of protein-bound amino acids, oxidative cleavage of the protein backbone, and incorporation of carbonyls from glycoxidation or lipoxidation.

    “Protein carbonylation is a stable modification induced by ROS via three pathways: direct oxidation of protein-bound amino acids, oxidative cleavage of the protein backbone, and incorporation of carbonyls from glycoxidation or lipoxidation [63].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [63] Dalle-Donne, 2003, Protein carbonylation in human diseases, Trends Mol. Med. source

  • May combine two claims.

    Premisereasoning

    Absolute quantification is problematic with immunochemical methods because there is no market-wide standardization of reference material, so concentration values may vary depending on the used kit.

    “Besides structural uncertainty, absolute quantification is problematic with immunochemical methods. Most commercial suppliers of immunoassays provide standards in their kits. Since there is no market wide standardization of reference material, concentration values may vary depending on the used kit.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    On low-resolution instruments, isobaric ions from non-modified peptides can yield false-positive results, so data evaluation should be treated with caution.

    “Especially on low resolution instruments, data evaluation should be treated with caution since isobaric ions from non-modified peptides can yield false-positive results [100].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [100] Abello, 2009, Protein tyrosine nitration: selectivity, physicochemical and biological consequences, denitration, and proteomics methods for the identification of tyrosine-nitrated proteins, J. Proteome Res. source

  • May combine two claims.

    Premisecitation

    CML is formed by oxidative degradation of fructoselysine (Amadori product) and alternatively by reaction of the α-dicarbonyl compound glyoxal with lysine via an isomerization mechanism.

    “CML was the first discovered glycoxidation product and is formed by oxidative degradation of fructoselysine (Amadori product) [35]. An alternative pathway is the reaction between the α-dicarbonyl compound glyoxal and lysine leading to CML formation via an isomerization mechanism [36].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [35] Ahmed, 1986, Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein, J. Biol. Chem. source
    • cites [36] Glomb, 1995, Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction, J. Biol. Chem. source

  • May combine two claims.

    Premisereasoning

    Most commercial antibodies lack full chemical and structural characterization and their target epitopes remain unknown; most antibodies detect different oxidation products due to poor antigen preparation and characterization.

    “However, most commercial antibodies lack this characterization and their target epitopes remain unknown. Most antibodies detect different oxidation products which can be explained by poor antigen preparation and characterization.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationnone given

    Fully targeted analysis of oxidative protein modifications is the strategy with the highest sensitivity and most accurate quantification.

    “Fully targeted analysis of oxidative protein modifications is the strategy with the highest sensitivity and most accurate quantification.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Nitric oxide readily reacts with superoxide anions forming reactive nitrogen species such as peroxynitrite and nitrogen dioxide, which are potent oxidizing agents in vivo.

    “Apart from its role in cell signaling •NO readily reacts with superoxide anions forming RNS such as peroxynitrite and nitrogen dioxide [71]. These RNS are potent oxidizing agents in vivo [72].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [71] White, 2010, Nitrosative modifications of protein and lipid signaling molecules by reactive nitrogen species, Am. J. Physiol. Endocrinol. Metab. source
    • cites [72] Radi, 2018, Oxygen radicals, nitric oxide, and peroxynitrite: redox pathways in molecular medicine, Proc. Natl. Acad. Sci. Unit. States Am. source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Carbonyl compounds can also be formed during lipid peroxidation and the Maillard reaction, leading to an overestimation of protein oxidation.

    “It is important to notice that carbonyl compounds can also be formed during lipid peroxidation and the Maillard reaction, leading to an overestimation of protein oxidation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Quantification with standard material for oxidative modifications requires synthesis efforts or cannot always be implemented.

    “However, since standard material is often not available, especially not in peptide form, this approach requires synthesis efforts or cannot always be implemented.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Standard material for oxidative modifications is often not available, especially in peptide form.

  • May combine two claims.

    Premisecitation

    The DNPH method is unspecific towards other oxidation products besides carbonyl groups; sulfenic acids, which are not of carbonyl form, still react in the assay.

    “An important drawback of the DNPH method is its unspecific nature towards other oxidation products besides carbonyl groups. It was mentioned by Hellwig [84] that sulfenic acids, which are not of carbonyl form, still react in the assay.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [84] Hellwig, 2019, The chemistry of protein oxidation in food, Angew Chem. Int. Ed. Engl. source

  • May combine two claims.

    Premisecitation

    Oxidative cleavage of the peptide backbone is initiated by superoxide-mediated alkoxyl radical formation at the α-carbon next to a peptide bond and proceeds via the diamide or α-amidation pathway.

    “Generally, oxidative cleavage of the peptide backbone is initiated by superoxide mediated alkoxyl radical formation at the α-carbon next to a peptide bond. The alkoxyl radical fragments either by the homolytic cleavage of the carbon-carbon bond (diamide pathway) or the carbon-nitrogen bond (α-amidation pathway) (Fig. 5C).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [66] Stringfellow, 2014, Selectivity in ROS-induced peptide backbone bond cleavage, J. Phys. Chem. source

  • May combine two claims.

    Premisereasoning

    Complete digestion of proteins down to individual modified amino acids followed by MS allows identification of new oxidation products and total quantification of a particular product, but lacks information on the specific protein modified or the exact site of modification.

    “A special case of the “bottom-up” approach is complete digestion of the protein down to individual (modified) amino acids followed by MS analysis. It was used to investigate a wide range of oxidized amino acids, thus, identifying new oxidation products and allowing total quantification of a particular product. A drawback of complete digestion is the lack of information on the specific protein that”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Formation of nitric oxide is catalyzed by three isoforms of nitric oxide synthase (endothelial, neuronal, inducible), all using arginine and oxygen to produce citrulline and nitric oxide.

    “Formation of •NO is catalyzed by three isoforms of nitric oxide synthase (NOS) (Fig. 6): endothelial NOS, neuronal NOS or inducible NOS. All NOS isoforms use arginine and oxygen to produce citrulline and •NO [70].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [70] Hess, 2005, Protein S-nitrosylation: purview and parameters, Nat. Rev. Mol. Cell Biol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premisenone given

    Mass spectrometry is currently the gold standard and arguably the most informative technique for protein analysis.

    “Compared to other methods available, MS is currently the gold standard and arguably the most informative technique for protein analysis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Neutralization with sodium hydroxide shifts the absorbance of protein-conjugated hydrazones from 370 nm to 450 nm, thereby eliminating interference with unbound DNPH.

    “Neutralization shifts the absorbance of protein-conjugated hydrazones from 370 nm to 450 nm, thereby eliminating the interference with unbound DNPH.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [83] Mesquita, 2014, Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins, Anal. Biochem. source

  • May combine two claims.

    Premisecitation

    Carboxymethyl lysine (CML) is the most prominent and one of the most abundant AGEs in vivo.

    “The most prominent and one of the most abundant AGEs in vivo is carboxymethyl lysine (CML), which was extensively reviewed before [[32], [33], [34]].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [[32], [33], [34]]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Electron capture dissociation (ECD) and electron transfer dissociation (ETD) are particularly good at preserving labile posttranslational protein modifications.

    “Dissociation methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) are particularly good at preserving labile posttranslational protein modifications.”
    Quote in the paperNot a substantial claim

  • May combine two claims. The quote only partly states this claim.

    Premisereasoning

    Fluorescence spectroscopy can serve as a sum parameter for overall protein oxidation, but has limited selectivity and no quantitative calibration can be achieved because several fluorophores contribute to total fluorescence.

    “Fluorescence spectroscopy can serve as a sum parameter for overall protein oxidation. It needs to be considered that several oxidative and also glycoxidative modifications of amino acid residues can contribute to fluorescence signals. The limited selectivity is a major drawback of fluorescence spectroscopy for studying protein oxidation products.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Formation of AGEs does not generally require oxidative conditions; only selected AGEs are generated by oxidation, called glycoxidation products.

    “It is important to note that the formation of AGEs does not generally require oxidative conditions and only selected AGEs are generated by oxidation. This special subset of AGEs is called glycoxidation products, because they are formed by a combination of glycation and oxidation (Fig. 3) [29].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [29] Lima, 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisenone given

    The accessibility and reactivity of carbonyl groups depends on the tertiary structure of proteins; protein crosslinking or pronounced tertiary structures can hide DNPH-active carbonyl structures inside proteins.

    “The accessibility and reactivity of carbonyl groups depends on the tertiary structure of proteins. Protein crosslinking or pronounced tertiary structures can hide DNPH-active carbonyl structures inside of proteins making them unavailable for detection.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationnone given

    Top-down MS remains attractive for evaluating the overall modification status of proteins, but because of its drawbacks, complementary bottom-up proteomics is needed for a comprehensive understanding of protein structure.

    “Taken together, top-down MS remains an attractive technique to evaluate the overall modification status of proteins, but due to its various drawbacks, complementary techniques, such as bottom-up proteomics, need to be employed to acquire a comprehensive understanding of the protein structure.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

    Amino acid analysis reveals modification site but not the type of modification.

    “A method which reveals modification site, but not the type of modification is amino acid analysis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    All of these studies are related to oxidation of free amino acids or glutathione and therefore cannot be considered studies investigating direct protein oxidation.

    “All of these studies are related to the oxidation of free amino acids or glutathione and can therefore not be considered as studies investigating direct protein oxidation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    HPLC/MS/MS approaches for detecting cysteine residues provide high sensitivity, specificity and reproducibility but are limited for routine clinical applications because of high equipment costs and low sample throughput.

    “In particular, the latter approaches have been used in recent human studies, providing high sensitivity, specificity and reproducibility, but are limited for routine clinical applications, because of high costs for laboratory equipment and low sample throughput.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    Identification of valid biomarkers of protein oxidation has indispensable relevance to improve understanding of human disease pathogenesis and to provide a tool to capture disease state and develop potential treatment strategies.

    “Therefore, the identification of valid biomarkers of protein oxidation has an indispensable relevance to improve the understanding of human disease pathogenesis and provide an important tool to capture disease state as well as develop potential treatment strategies.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Methods that monitor aggregate formation by gel electrophoresis or biophysical methods do not give information about the type or site of modification and are therefore only of limited use for biomarker studies.

    “However, these methods do not give any information about the type or site of modification and are therefore only of limited use for biomarker studies.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    For detailed information on modification products and the types of proteins, MS analysis of peptides is inevitable.

    “For detailed information on modification products and the types of proteins, MS analysis of peptides, as described above, is inevitable.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationnone given

    Quantitative data in amino acid analysis can easily be achieved using external calibration and, for MS analysis, internal isotope-labeled standards.

    “Quantitative data can easily be achieved by using external calibration and, in case of MS analysis, internal isotope-labeled standards.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Acid hydrolysis conditions lead to loss of cysteine, cystine, and some tryptophan species, whereas alkaline conditions preserve tryptophan species but break down arginine, serine, threonine and cysteine.

    “Acid hydrolysis conditions lead to a loss of cysteine, cystine, and some tryptophan species, whereas alkaline conditions preserve tryptophan species, but breaks down arginine, serine, threonine and cysteine [107].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [107] Fountoulakis, 1998, Hydrolysis and amino acid composition of proteins, J. Chromatogr. A source

  • May combine two claims.

    Premisecitation

    Farhan et al. found elevated AGE levels comparing T2D patients with and without vascular diseases using a competitive ELISA Kit without specification of detected glycoxidation products.

    “In contrast, Farhan et al. found elevated AGE levels comparing the latter groups by using a competitive ELISA Kit without specification of detected glycoxidation products [159,160].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [159] Farhan, 2019, Advanced glycation end products (AGEs) and their soluble receptors (sRAGE) as early predictors of reno-vascular complications in patients with uncontrolled type 2 diabetes mellitus, Diabetes Metab Syndr source

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    It is important to discriminate and selectively analyze the various structures formed by glycoxidation reactions.

    “This emphasizes the importance of discrimination and selective analysis of the various structures formed by glycoxidation reactions”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The TBARS assay is unspecific and measures total oxidative stress rather than MDA alone.

    “This approach is the method of choice for the quantification of free MDA due to simplicity and low costs, but is often criticized as being unspecific and rather measuring total oxidative stress instead of MDA alone [181,182].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [181] Niki, 2014, Biomarkers of lipid peroxidation in clinical material, Biochim. Biophys. Acta source
    • cites [182] Grotto, 2007, Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography-visible detection, J. Pharmaceut. Biomed. Anal. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Further methods such as HPLC with UV or fluorescence detection have been developed to improve specificity of MDA measurement.

    “Beside this assay, further methods have been developed in order to improve the specificity of MDA measurement, such as HPLC combined with UV or fluorescence detection [182,184].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [182] Grotto, 2007, Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography-visible detection, J. Pharmaceut. Biomed. Anal. source
    • cites [184] Domijan, 2015, Quantification of malondialdehyde by HPLC-FL - application to various biological samples, Biomed. Chromatogr. source

  • May combine two claims.

    Interpretationreasoning

    Protein carbonyl content can be seen as a biomarker of global oxidative protein damage, with the advantage of early formation and long circulation periods compared to other parameters of oxidative stress.

    “protein carbonyl content can be seen as a biomarker of global oxidative protein damage, with the advantage of early formation and long circulation periods compared to other parameters of oxidative stress”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Elevated levels of protein carbonyls have been observed in several human diseases and are associated with the aging process.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    4-HNE and MDA presumably correlate with the amount of protein modifications formed by reaction with this carbonyl species.

    “4-HNE and MDA, which presumably correlate with the amount of protein modifications formed by reaction with this carbonyl species”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationnone given

    Protein oxidation seems to play a critical role in several pathologies and in the aging process.

    “protein oxidation seems to play a critical role in several pathologies and in the aging process”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Weber et al. provided a simple and fast indirect ELISA method for measurement of protein-bound 3-NT in human serum and plasma samples.

    “Weber et al. provided a simple and fast indirect ELISA method for the measurement of protein-bound 3-NT in human serum and plasma samples.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [196] Weber, 2012, Rapid and sensitive determination of protein-nitrotyrosine by ELISA: application to human plasma, Free Radic. Res. source

  • May combine two claims.

    Premisecitation

    Several limitations of ELISA for 3-NT have been highlighted in the literature, including low sensitivity, minor specificity of applied antibodies, susceptibility to errors, and missing detection of free 3-NT.

    “several limitations have been highlighted in the literature, such as low sensitivity, minor specificity of applied antibodies, the susceptibility to errors, or missing detection of free 3-NT”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [196] Weber, 2012, Rapid and sensitive determination of protein-nitrotyrosine by ELISA: application to human plasma, Free Radic. Res. source
    • cites [197] Rabbani, 2008, Assay of 3‐nitrotyrosine in tissues and body fluids by liquid chromatography with tandem mass spectrometric detection source
    • cites [198] Tsikas, 2014, Mass spectrometry and 3-nitrotyrosine: strategies, controversies, and our current perspective, Mass Spectrom. Rev. source

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Drawing a final conclusion on glycoxidation products as relevant biomarkers of protein oxidation is hardly possible based on the included studies published in the last years.

    “drawing a final conclusion on glycoxidation products as relevant biomarkers of protein oxidation is hardly possible based on the included studies published in the last years”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The most accurate detection of 3-NT requires the combination of LC with MS/MS, providing the opportunity to distinguish between free, protein-bound, or total 3-NT.

    “The most accurate detection of 3-NT, however, requires the combination of LC with MS/MS providing the opportunity to distinguish between free, protein-bound, or total 3-NT”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [198] Tsikas, 2014, Mass spectrometry and 3-nitrotyrosine: strategies, controversies, and our current perspective, Mass Spectrom. Rev. source

  • May combine two claims.

    Interpretationreasoning

    Despite partially inconsistent results, elevated 3-NT levels are associated with multiple diseases and aging, indicating its potential as biomarker for protein oxidation in clinical research.

    “Despite partially inconsistent results in studies of recent years, elevated 3-NT levels are associated with multiple diseases and aging, indicating its potential as biomarker for protein oxidation in clinical research.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    These protein modifications are generally more stable compared to their reactive precursors and are important biomarkers for monitoring medium- to long-term oxidative and nitrosative stress.

    “These modifications are generally more stable compared to their reactive precursors and, thus, important biomarkers for monitoring medium-to long-term oxidative and nitrosative stress.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationnone given

    Among the presented methods, MS is the most accurate and informative technique, capable of resolving remaining questions regarding protein modifications.

    “Among the presented methods, MS is certainly the most accurate and informative technique, capable of resolving remaining questions regarding protein modifications.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: MS approaches are considered the gold standard for 3-NT measurement.

  • May combine two claims.

    Premisecitation

    The Weber et al. ELISA method has high detection limits, small sample volumes, and opportunity for high throughput screening, making it useful for clinical applications.

    “High detection limits, small sample volumes, and the opportunity for high throughput-screening makes this method useful for clinical applications”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [196] Weber, 2012, Rapid and sensitive determination of protein-nitrotyrosine by ELISA: application to human plasma, Free Radic. Res. source

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    As a result of oxidative and nitrosative stress, an incredible number of distinct protein modifications is formed.

    “As a result, an incredible number of distinct protein modifications is formed.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Impaired balance between generation and degradation of reactive oxygen and nitrogen species in aging and disease causes oxidative and nitrosative stress.
76 of 76

  • The authors state this as an assumption.

    Proteins accumulate evidence of oxidative damage in relation to disease, although some associations are only associations and oxidation is not always causal but a contributing factor.

    “It can be therefore assumed that proteins also accumulate evidence of oxidative damage in relation to disease, although some of these are only associations and oxidation is not always causal, but a contributing factor.”

  • The paper relies on this without support.

    To function as suitable biomarkers, protein oxidation products must be stable, accumulate in detectable concentrations, and correlate with disease severity.

    “To function as suitable biomarkers, it is critical that protein oxidation products are stable, accumulate in detectable concentrations, and correlate with disease severity.”

  • The paper relies on this without support.

    Sample availability is an important factor limiting the reliability of a biomarker.

    “Sample availability is another important factor limiting the reliability of a biomarker.”

  • The paper relies on this without support.

    Unmodified reference material is commonly not available in biological samples.

    “A major challenge in analyzing protein modifications in biological samples is that unmodified reference material is commonly not available.”

  • The paper relies on this without support.

    Modified peptides often occur only at low abundances, making it unlikely that untargeted approaches cover them.

    “However, modified peptides often occur only at low abundancies making it unlikely that they are covered by untargeted approaches.”

  • The paper relies on this without support.

    Standard material for oxidative modifications is often not available, especially in peptide form.

    “However, since standard material is often not available, especially not in peptide form, this approach requires synthesis efforts or cannot always be implemented.”

  • The paper relies on this without support.

    MDA-modified proteins have not been used as biomarkers in recent clinical investigations.

    “To our knowledge, MDA modified proteins have not been used as biomarkers in recent clinical investigations although antibodies are commercially available and highly selective MS methods have been established, previously [57].”

  • The paper relies on this without support.

    Only Weber et al. has determined 3-NT as a function of age in three different cohorts of the MARK-AGE study.

    “To our knowledge, only Weber et al. determined 3-NT as a function of age in three different cohorts of the MARK-AGE study [194].”

  • The paper relies on this without support.

    3-NT can be measured in plasma, serum, and tissue samples by methods including IHC, ELISA, HPLC, or LC-MS/MS.

    “The measurement of 3-NT can be performed in plasma, serum as well as tissue samples by different methods, including IHC, ELISA, HPLC or the combination of LC with MS/MS”

  • The paper relies on this without support.

    MS approaches are considered the gold standard for 3-NT measurement.

    “MS approaches have been considered as gold standard”

  • The paper relies on this without support.

    Commercially available ELISAs are preferentially used for 3-NT quantification in clinical studies due to standardization, easy sample preparation, need of conventional equipment, and high sample throughput.

    “commercially available ELISAs are preferentially used for 3-NT quantification in clinical studies due to standardization, easy sample preparation, the need of conventional equipment, and high sample throughput”

  • The paper relies on this without support.

    Reactive oxygen and nitrogen species are ubiquitous side-products of metabolism.

    “Reactive oxygen and nitrogen species are ubiquitous side-products of metabolism.”

  • The paper relies on this without support.

    Impaired balance between generation and degradation of reactive oxygen and nitrogen species in aging and disease causes oxidative and nitrosative stress.

    “Impaired balance between their generation and degradation in aging and disease causes oxidative and nitrosative stress.”

  • The paper relies on this without support.

    Many biological disciplines use immunoassays as routine methods.

    “Many biological disciplines use immunoassays as routine methods.”

  • The paper relies on this without support.

    Immunological methods are often based on monoclonal antibodies or polyclonal antisera obtained after immunization with immunogens, lacking full chemical or structural characterization.

    “Immunological methods are often based on monoclonal antibodies or polyclonal antisera obtained after immunization with immunogens, lacking full chemical or structural characterization.”

  • The paper relies on this without support.

    In many cases, the epitopes recognized by the antibodies remain unknown.

    “In many cases, the epitopes recognized by the antibodies remain unknown.”

  • The paper relies on this without support.

    Recent studies on advanced lipoxidation endproducts mainly focused on the reactive intermediates 4-HNE and MDA.

    “recent studies mainly focused on the reactive intermediates 4-HNE and MDA”

  • The paper relies on this without support.

    Elevated levels of protein carbonyls have been observed in several human diseases and are associated with the aging process.

    “elevated levels of protein carbonyls have been observed in several human diseases and are associated with the aging process”
18 of 18

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Glaucoma study specimen changes from serum to plasma

    Minor

    The glaucoma row lists serum and aqueous humor as specimens, but its outcome says the elevated levels were measured in plasma and aqueous humor.

  • Reference 85 does not match the attributed method

    Minor

    The text attributes a 2019 carbonyl-detection method to Ma and colleagues and cites reference 85, but reference 85 lists Swiderska and colleagues and concerns oxidative-stress biomarkers in NAFLD.

  • Pacher review year differs from its reference

    Minor

    The text dates the cited Pacher review to 2008, while reference 188 gives its publication year as 2007.

  • Cysteine study is called non-protein oxidation despite protein-bound outcome

    Minor

    The narrative says all preceding cysteine studies examined free amino acids or glutathione rather than direct protein oxidation. The preceding table, however, describes the sickle-cell disease and sepsis outcome as protein-bound cysteine.

4 of 4

Cites

Flagged first, then by citations

2 of the 251 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Kehm, R., Baldensperger, T., Raupbach, J., & Höhn, A.
Affiliations
  • German Institute of Human Nutrition (DE)
  • German Center for Diabetes Research (DE)
Corresponding author
Höhn, Annika
ORCID
Venue
Redox Biology
Publisher
Elsevier BV
Published
2021-06
Volume
42
Pages
101901
ISSN
2213-2317 (ISSN-L)
DOI
10.1016/j.redox.2021.101901
PMC
PMC8113053
OpenAlex
W3129842026
Semantic Scholar
e11c1d0fd97bbda9ef5a77276a54fc8d2150ae90
Cited by
348
References
251
Citations by year
2026: 80 · 2025: 92 · 2024: 74 · 2023: 58 · 2022: 31 · 2021: 13
FWCI
11.78
Type
journal article
Language
en
Open access
Licence
Locations
  • Redox Biology (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Informationszentrum Lebenswissenschaften (ZB MED) (submittedVersion, cc-by-nc-nd)
Funders
Bundesministerium für Bildung und Forschung
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Health Sciences › Medicine › Biochemistry › Antioxidant Activity and Oxidative Stress
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Advanced Glycation End Products research
Keywords
protein oxidation, reactive oxygen species, oxidative stress, glycoxidation, lipid oxidation, post-translational modification, redox signaling, oxidative damage biomarkers, aging, disease
MeSH terms
Humans, Oxidation-Reduction, Proteins, Biomarkers, Reactive Oxygen Species, Oxidative Stress

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-18.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-18.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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