Topographical distribution of phosphorylation sites of phosvitins by mass spectrometry

Aim Application of quantitative stable-isotope-labeling chemistries and mass spectrometry (MS) to determine alterations in gingival crevicular fluid (GCF) proteome in periodontal disease. Materials and Methods Quantitative proteome of GCF from 40 healthy individuals versus 40 patients with periodontal disease was established using 320 GCF samples and stable-isotope-labeling reagents, ICAT and mTRAQ, with MS technology and validated by enzyme-linked immunosorbent methods. Results We have identified 238 distinct proteins of which 180 were quantified in GCF of both healthy and periodontal patients with additional 26 and 32 distinct proteins that were found only in GCF of healthy or periodontal patients. In addition, 42 pathogenic bacterial proteins and 11 yeast proteins were quantified. The data highlighted a series of proteins not quantified previously by large-scale MS approaches in GCF with relevance to periodontal disease, such as host derived Ig alpha-2 chain C, Kallikrein-4, S100-A9, transmembrane proteinase 13, peptidase S1 domain, several collagen types and pathogenic bacterial proteins e.g., formamidase, leucine amidopeptidase and virulence factor OMP85. Conclusions The innovative analytical approaches provided detailed novel changes in both host and microbial derived GCF proteomes of periodontal patients. The study defined 50 host and 16 pathogenic bacterial proteins significantly elevated in periodontal disease most of which were novel with significant potential for application in the clinical arena of periodontal disease.

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“…, Czernick et al. ). Figure Panel 3A,B show typical LC‐ESI‐MS/MS relative base‐peak ion abundance for ICAT and mTRAQ labelled GCF proteins.…”

Section: Methodsmentioning

confidence: 98%

Quantitative gingival crevicular fluid proteome in health and periodontal disease using stable isotope chemistries and mass spectrometry

Carneiro

Nouh

Salih

2014

J Clinic Periodontology

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“…These residues do not account for all the phosphate incorporated into phosvitin 2, but they are sufficient to conclude that, based on the absence of a canonical Fam20C consensus site (S-x-E), none of the identified phosphoserines is expected to be generated by Fam20C. This applies in particular to the stretch of 12 adjacent serines, 11 of which are phosphorylated, that correspond to the [15]; the sequence encompassing R 3 S 12 R 3 peptide is underlined. (B) Phosvitin derived from zebrafish VTG1.…”

Section: Resultsmentioning

confidence: 94%

“…A. Highlighted are 57 p‐Ser sites unambiguously localized by MS/MS sequence analyses . These residues do not account for all the phosphate incorporated into phosvitin 2, but they are sufficient to conclude that, based on the absence of a canonical Fam20C consensus site (S‐x‐E), none of the identified phosphoserines is expected to be generated by Fam20C.…”

Section: Resultsmentioning

confidence: 99%

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The Golgi ‘casein kinase’ Fam20C is a genuine ‘phosvitin kinase’ and phosphorylates polyserine stretches devoid of the canonical consensus

et al. 2018

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Egg yolk phosvitins, generated through the fragmentation of vitellogenins, are among the most heavily phosphorylated proteins ever described. Despite the early discovery in 1900 that chicken phosvitin is a phosphoprotein and its subsequent employment as an artificial substrate for a number of protein kinases, the identity of the enzyme(s) responsible for its phosphorylation remained a matter of conjecture until present. Here we provide evidence that phosvitin phosphorylation is catalyzed by Fam20C, an atypical protein kinase recently identified as the genuine casein kinase and responsible for the phosphorylation of many other secreted proteins at residues specified by the S-x-E/pS consensus. Such a conclusion is grounded on the following observations: i) the levels of Fam20C and phosphorylated vitellogenin rise in parallel upon treatment of zebrafish with oestrogens; ii) Zebrafish phosvitin is readily phosphorylated upon coexpression in U2OS cells with Fam20C, but not with its catalytically inactive mutant; iii) a peptide reproducing a stretch of 12 serines, which are phosphorylated in chicken phosvitin despite lacking the C terminal priming motif S-x-E, is efficiently phosphorylated by both recombinant and native Fam20C. The last finding expands the repertoire of potential targets of Fam20C to include several proteins known to harbour (p-Ser)n clusters not specified by any known kinase consensus.

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“…A detailed liquid chromatography/tandem mass spectrometry approach for phosphoproteomics has been previously described. [31][32][33] Mass spectrometry was performed in primary human and renal cells at baseline and after simulated ischemic stress, in fresh human and murine kidney tissue homogenates before and after frank ischemia, and in human and murine urine from patients without AKI and patients with AKI before and after experimental AKI, respectively. At least three replicates of each purified NPM sample harvested from renal cells, kidney tissue, and fresh urine were subjected to mass spectrometry.…”

Section: Mapping Of Npm Phosphorylation Sites By Massmentioning

confidence: 99%

Nucleophosmin Phosphorylation as a Diagnostic and Therapeutic Target for Ischemic AKI

Wang

Salih

Igwebuike

et al. 2018

JASN

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Background Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Differential nucleophosmin (NPM) phosphorylation is a potential early marker of ischemic renal cell injury and a therapeutic target. Methods Differential NPM phosphorylation was assessed by mass spectrometry in NPM harvested from murine and human primary renal epithelial cells, fresh kidney tissue, and urine before and after ischemic injury. The biologic behavior and toxicity of NPM was assessed using phospho-NPM mutant proteins that either mimic stress-induced or normal NPM phosphorylation. Peptides designed to interfere with NPM function were used to explore NPM as a therapeutic target. Results Within hours of stress, virtually identical phosphorylation changes were detected at distinct serine/threonine sites in NPM harvested from primary renal cells, tissue, and urine. A phosphomimic NPM protein that replicated phosphorylation under stress localized to the cytosol, formed monomers that interacted with Bax, a cell death protein, coaccumulated with Bax in isolated mitochondria, and significantly increased cell death after stress; wild-type NPM or a phosphomimic NPM with a normal phosphorylation configuration did not. Three renal targeted peptides designed to interfere with NPM at distinct functional sites significantly protected against cell death, and a single dose of one peptide administered several hours after ischemia that would be lethal in untreated mice significantly reduced AKI severity and improved survival. Conclusions These findings establish phosphorylated NPM as a potential early marker of ischemic AKI that links early diagnosis with effective therapeutic interventions.

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Topographical distribution of phosphorylation sites of phosvitins by mass spectrometry

Cited by 13 publications

References 49 publications

Quantitative gingival crevicular fluid proteome in health and periodontal disease using stable isotope chemistries and mass spectrometry

Quantitative gingival crevicular fluid proteome in health and periodontal disease using stable isotope chemistries and mass spectrometry

The Golgi ‘casein kinase’ Fam20C is a genuine ‘phosvitin kinase’ and phosphorylates polyserine stretches devoid of the canonical consensus

Nucleophosmin Phosphorylation as a Diagnostic and Therapeutic Target for Ischemic AKI

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