Seeing Citrulline: Development of a Phenylglyoxal-Based Probe To Visualize Protein Citrullination

Bicker, Kevin L.; Subramanian, Venkataraman; Chumanevich, Alexander A.; Hofseth, Lorne J.; Thompson, Paul R.

doi:10.1021/ja308871v

Cited by 97 publications

(144 citation statements)

References 28 publications

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“…Phenylglyoxal-rhodamine Probe-Another method that was recently described to be applicable for the detection of citrullinated proteins is based on the chemical reaction of the ureido group of citrulline with phenylglyoxal (36,37). Phenylglyoxal has been reported to react specifically with arginine residues under neutral or basic conditions and with citrulline residues under highly acidic conditions.…”

Section: Methods For the Detection Of Citrullinated Proteins On Westementioning

confidence: 99%

Methods for the Detection of Peptidylarginine Deiminase (PAD) Activity and Protein Citrullination

Hensen¹,

Pruijn²

2014

Molecular & Cellular Proteomics

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The post-translational conversion of peptidylarginine to peptidylcitrulline, a process also known as citrullination, is catalyzed by the enzyme family of peptidylarginine deiminases (PADs) and has been demonstrated to be involved in many physiological processes, including the regulation of gene expression. In addition, citrullination has been shown to be associated with several diseases, such as cancer, multiple sclerosis, rheumatoid arthritis, and Alzheimer's disease. To get more insight into the role of PAD enzymes and citrullination in both health and disease, experimental strategies to study PAD activity and to characterize citrullinated proteins in complex biological samples are crucial. Here, we describe the chemical, proteomic and antibody-based procedures that are currently available and discuss their applicability for the analysis of complex samples. The methods that have been developed can be used to provide more insight in the substrate specificity of PAD enzymes. Because the evidence that PADs play a pathophysiological role in the diseases mentioned above is increasing, they become attractive targets for therapeutic interventions. More knowledge of PAD specificity and the availability of reliable, high-throughput assays for PAD activity will facilitate the development of highly specific PAD inhibitors. Molecular & Cellular Proteomics 13:

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Section: Methods For the Detection Of Citrullinated Proteins On Westementioning

confidence: 99%

Methods for the Detection of Peptidylarginine Deiminase (PAD) Activity and Protein Citrullination

Hensen¹,

Pruijn²

2014

Molecular & Cellular Proteomics

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“…Further, deamidation of Gln or Asn produces mass shifts identical to citrullination. Several chemical proteomic strategies aid in the identification of citrullinated peptides (22)(23)(24)(25), but citrulline site identifications particularly (and PTM site identifications generally) require observation of an ion series containing Significance Protein posttranslational modifications (PTMs) regulate virtually every aspect of cellular function. Mapping PTMs is a key application of protein mass spectrometry (MS), which analyzes thousands of peptides generated in enzymatic protein digests.…”

mentioning

confidence: 99%

Evolution of a mass spectrometry-grade protease with PTM-directed specificity

Tran

Cavett

Dang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (k cat /K M = 6.9 × 10 5 M). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y 1 ion corresponding to citrulline (176.1030 m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.T he identification and mapping of protein posttranslational modifications (PTMs), which underpin the regulation of virtually every cellular function, represents one of the most significant challenges of modern-day analytical science. Accessing this biochemical information requires direct observation of the modified site, often in low abundance within the complex cellular milieu. The ability to address this sample complexity is precisely why tandem mass spectrometry (MS/MS) now dominates proteomic analysis. LC-MS/MS instruments can analyze mixtures of thousands of peptides, acquiring the mass-to-charge ratio (m/z) of each peptide precursor ion (MS1) and fragmenting the most abundant precursors for a second dimension of MS analysis (MS2). Database searching of MS/MS "bottom-up" proteomic data entails matching observed precursor and fragmentation product ion masses with those of a theoretical protein digest, returning the amino acid sequence of each ion (including any PTMs) and statistical confidence of the sequence match (1, 2).Bottom-up MS/MS workflows generate peptides for analysis almost ubiquitously by digestion using trypsin, which cleaves the C-terminal peptide bonds of Arg and Lys with high catalytic efficiency and selectivity (3). Regions of a protein too densely or too sparsely populated with tryptic sites result in sequencing coverage gaps, which can conceal PTM sites during mapping experiments. Digestion with alternative proteases (4, 5), which reinforce tryptic cleavage...

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“…Representative Comets for each group are shown. protein citrullination in mice fed Cl-amidine (Bicker et al, 2012b), suppresses colitis (Fig. 2).…”

Section: Discussionmentioning

confidence: 91%

“…Similarly, Cl-amidine increased weight gain compared with AOM 1 DSS-treated mice. It is important to note that under such conditions Clamidine does suppress protein citrullination (Bicker et al, 2012b).…”

Section: Resultsmentioning

confidence: 99%

Inhibiting Protein Arginine Deiminases Has Antioxidant Consequences

Witalison

Cui

Hofseth

et al. 2015

J Pharmacol Exp Ther

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Ulcerative colitis is a dynamic, idiopathic, chronic inflammatory condition that carries a high colon cancer risk. We previously showed that Cl-amidine, a small-molecule inhibitor of the protein arginine deiminases, suppresses colitis in mice. Because colitis is defined as inflammation of the colon associated with infiltration of white blood cells that release free radicals and citrullination is an inflammationdependent process, we asked whether Cl-amidine has antioxidant properties. Here we show that colitis induced with azoxymethane via intraperitoneal injection 1 2% dextran sulfate sodium in the drinking water is suppressed by Cl-amidine (also given in the drinking water).Inducible nitric oxide synthase, an inflammatory marker, was also downregulated in macrophages by Cl-amidine. Because epithelial cell DNA damage associated with colitis is at least in part a result of an oxidative burst from overactive leukocytes, we tested the hypothesis that Cl-amidine can inhibit leukocyte activation, as well as subsequent target epithelial cell DNA damage in vitro and in vivo. Results are consistent with this hypothesis, and because DNA damage is a procancerous mechanism, our data predict that Clamidine will not only suppress colitis, but we hypothesize that it may prevent colon cancer associated with colitis.

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Seeing Citrulline: Development of a Phenylglyoxal-Based Probe To Visualize Protein Citrullination

Cited by 97 publications

References 28 publications

Methods for the Detection of Peptidylarginine Deiminase (PAD) Activity and Protein Citrullination

Methods for the Detection of Peptidylarginine Deiminase (PAD) Activity and Protein Citrullination

Evolution of a mass spectrometry-grade protease with PTM-directed specificity

Inhibiting Protein Arginine Deiminases Has Antioxidant Consequences

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