High pressure liquid chromatography of fibrinopeptides derived from eight mammalian fibrinogens

Sellers, J.P.; Clark, Haydn

doi:10.1016/0049-3848(81)90241-3

Cited by 10 publications

(6 citation statements)

References 9 publications

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“…Peptide reference materials of bovine fibrinopeptides A and B were prepared (synthesised to ≥95% purity) by Severn Biotech Ltd. according to sequences published in NCBI Genbank (Table 1). While developing the method from that of Sellers and Clark,2 it was discovered that the low pH conditions used during extraction and analysis were causing the N‐terminal glutamine (Q) of bovine fibrinopeptide B to undergo a conversion into pyroglutamic acid (pE). This pE form of fibrinopeptide B was thus synthesised and used as a reference material throughout the analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Extraction of fibrinopeptides was performed at low pH in a modified method of that of Sellers and Clark2 which involves trichloroacetic acid (TCA) precipitation of the peptides. Meat or cooking liquor (2 g) was homogenised with 6 mL chilled 6.2% (w/v) TCA and precipitated on ice.…”

Section: Methodsmentioning

confidence: 99%

“…Fibrinogen is cleaved by the blood protease thrombin, which first removes a pair of peptides from the N‐terminus of the α ‐chains (fibrinopeptides A), then a second pair of peptides from the N‐terminus of the β ‐chains (fibrinopeptides B) 1. Fibrinopeptides A and B are hyper‐variable between species 2, 3…”

Section: Sequence Of Bovine Fibrinopeptides a And B Used To Prepare Smentioning

confidence: 99%

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Screening method for the addition of bovine blood‐based binding agents to food using liquid chromatography triple quadrupole mass spectrometry

Grundy¹,

Reece²,

Sykes³

et al. 2007

Rapid Comm Mass Spectrometry

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We report the development of a qualitative method to detect the addition of blood-based binding agents to food products. The method is based on the detection of species-specific marker peptides, fibrinopeptides, released from the blood protein fibrinogen during gelling of the blood protein by thrombin. The fibrinopeptides were isolated from foods spiked with commercial bovine binding agent by acid precipitation followed by enrichment using solid-phase extraction and analysed by liquid chromatography electrospray ionisation triple quadrupole mass spectrometry. Fibrinopeptide A was found to be an effective marker in fresh, processed and cooked food matrices spiked with 5% (v/w) bovine binding agent.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Sequence Of Bovine Fibrinopeptides a And B Used To Prepare Smentioning

confidence: 99%

See 1 more Smart Citation

Screening method for the addition of bovine blood‐based binding agents to food using liquid chromatography triple quadrupole mass spectrometry

Grundy¹,

Reece²,

Sykes³

et al. 2007

Rapid Comm Mass Spectrometry

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“…These samples were incubated with thrombin (2.5 U/ml) at 37°C for 60 min, and the reactions were terminated by addition of TCA to 5%. The samples were extracted with ether (Sellers and Clark, 1982), diluted 10-fold in 50 mMTris pH 7.4, 0.15 MNaCl, and assayed using a RIA Quant kit for human FPA (Mallinckrodt Diagnostics). Using antisera prepared against human FPA, this kit measures competition between the unknown peptide sample and 125I-labeled FPA compared to a standard curve from 1-40 ng/ml of FPA.…”

Section: Plasmid Constructions Resultsmentioning

confidence: 99%

Expression of a Cloned Human Fibrinogen cDNA inEscherichia coli: Synthesis of an A Alpha Polypeptide

Lord¹

1985

DNA

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The construction of a plasmid, p166.9, for the controlled synthesis of the A alpha-chain of human fibrinogen in Escherichia coli is described. The plasmid combines the tac promoter, constructed for controlled high-level peptide expression, with the promoter and signal peptide codons from an E. coli plasmid beta-lactamase gene and a cDNA of the A alpha-chain of human fibrinogen. The tac promoter is repressed in lacIQ strains of E. coli and induced by isopropylthio-beta-D-galactoside (IPTG). Protein blot analysis of lysates of cells carrying p166.9 demonstrates the IPTG-dependent synthesis of polypeptides which cross react with antisera to the A alpha-chain of human fibrinogen. The largest and predominant species corresponds to an apparent molecular weight of 63,000. When the cell growth media or cell lysates are treated with thrombin, the enzyme which normally releases fibrinopeptide A (FPA) from the A alpha-chain, FPA-like peptides are detectable by radioimmunoassay with antiserum prepared against human FPA. Thrombin-treated cell growth media prepared 4 hr after IPTG induction contained 340 ng/ml of FPA-like material; using a mass ratio of 40 for FPA to A alpha, this indicates that the A alpha-peptide concentration in the culture media is 13 micrograms/ml.

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“…The recent development of HPLC techniques for the analysis of fibrinopeptides Koehn & Canfield, 1981;Martinelli & Scheraga, 1979;Sellers & Clark, 1981) allows the isolation of these peptides from small amounts of material. The quantitative isolation and Correspondence: Dr C. Southan, Max-Planck-Institute for Biochemistry, Martinsried, Munich D-803 3 , German Federal Republic.…”

mentioning

confidence: 99%

Fibrinogen Manchester: identification of an abnormal fibrinopeptide A with a C‐terminal arginine→histidine substitution

et al. 1983

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Purified samples of fibrinogen Manchester, a congenital dysfibrinogenaemia with impaired fibrinopeptide A (FPA) release, were digested with thrombin. Amino acid sequencing of the fibrin showed that FPA had been completely released. High performance liquid chromatographic (HPLC) analysis of the clot supernatant showed the presence of a new peptide eluting ahead of the normal FPA. The amino acid composition and sequence of the new peptide established its identity as a variant of FPA containing histidine in position 16 instead of the usual arginine. The chromatograms from both siblings with the defect demonstrated that they were heterozygous for this clotting defect.

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High pressure liquid chromatography of fibrinopeptides derived from eight mammalian fibrinogens

Cited by 10 publications

References 9 publications

Screening method for the addition of bovine blood‐based binding agents to food using liquid chromatography triple quadrupole mass spectrometry

Screening method for the addition of bovine blood‐based binding agents to food using liquid chromatography triple quadrupole mass spectrometry

Expression of a Cloned Human Fibrinogen cDNA inEscherichia coli: Synthesis of an A Alpha Polypeptide

Fibrinogen Manchester: identification of an abnormal fibrinopeptide A with a C‐terminal arginine→histidine substitution

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