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W J Griffiths

Publications and source records attributed to W J Griffiths.

At least 19 recordsLinked to original sources
PubMed2006

Proteomic analysis of cytochromes P450: a mass spectrometry approach.

In human, the CYP (cytochrome P450) superfamily comprises 57 genes arranged in 18 families and 42 subfamiles. These genes encode for enzymes involved in the metabolism of drugs, foreign chemicals, fatty acids, eicosanoids and cholesterol. Additionally, they play roles in bile acid biosynthesis, steroid synthesis and metabolism, and vitamin D(3) synthesis and metabolism. Mutations in many CYP genes cause inborn errors of metabolism and contribute to increased risk of cancer. MS provides a convenient method for the identification and quantification of CYP enzymes, and in the present paper we will review the current state of the technology for such an analysis.

Amino Acid Sequence↗
PubMed2004

Identification of cytochrome P450 enzymes in human colorectal metastases and the surrounding liver: a proteomic approach.

We describe the direct identification of multiple cytochrome P450 (CYP) enzymes in healthy and cancerous tissue. CYPs in human liver colorectal metastases were compared with those in the surrounding liver using a mass spectrometry-based proteomic approach. Nano-scale reversed phase liquid chromatography combined with electrospray ionisation tandem mass spectrometry has been used to identify CYPs with no pre-selection of the proteins anticipated. Fourteen distinct CYP enzymes from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, 8B and 27A were positively identified; 13 in the liver samples and 12 in the tumour tissue. It was found that three of the colorectal metastases expressed essentially the same drug-metabolising pattern of CYPs as the surrounding liver, whilst three tumour samples from different individuals showed no CYP expression. This was likely in at least one case to be due to low sample mass. The CYP expression profile in an individual tumour is likely to be an important determinant in predicting the outcome of cancer chemotherapy.

Adult↗
PubMed2004

A proteomic approach to the identification of cytochrome P450 isoforms in male and female rat liver by nanoscale liquid chromatography-electrospray ionization-tandem mass spectrometry.

Nanoscale reversed-phase liquid chromatography (LC) combined with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used as a method for the direct identification of multiple cytochrome P450 (P450) isoforms found in male and female rat liver. In this targeted proteomic approach, rat liver microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel tryptic digestion of the proteins present in the 48- to 62-kDa bands. The resultant peptides were extracted and analyzed by LC-ESI-MS/MS. P450 identifications were made by searching the MS/MS data against a rat protein database containing 21,576 entries including 47 P450s using Sequest software (Thermo Electron, Hemel Hempstead, UK). Twenty-four P450 isoforms from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, CYP17, and CYP19 were positively identified in rat liver.

Amino Acid Sequence↗
PubMed2002

Characterization of carrot pectin methylesterase.

The most alkaline form of pectin methylesterase was purified from ripe carrot roots and used for structural analysis. Determination of an N-terminal blocking group and of the primary structure allowed comparisons with other forms, and facilitated crystallographic determination of the three-dimensional structure. The mature enzyme has 319 residues and the N-terminal blocking group was shown to be a pyroglutamyl residue derived from a glutaminyl cyclization. Few other methylesterases have been isolated and assigned to exact mature forms, and together with the present enzyme, only two have been analyzed in three-dimensional structure. However, comparison of 39 forms, mainly from GenBank data, reveals clear relationships and identifies subgroups of this enzyme type, deviating in structure but centering around two functionally important and conserved Asp residues at positions 136 and 157 in the carrot enzyme.

Amino Acid Sequence↗
PubMed2001

The palmitoyl groups of lung surfactant protein C reduce unfolding into a fibrillogenic intermediate.

Lung surfactant protein C (SP-C) is a lipophilic peptide that converts from a monomeric alpha-helical state into beta-sheet conformation and forms amyloid fibrils, a process which appears to be accelerated by removal of its two S-palmitoyl groups, and elevated amounts of non-palmitoylated SP-C are found in pulmonary alveolar proteinosis. Here, we used mass spectrometry to study the first step in fibrillogenesis of di-, mono- and non-palmitoylated SP-C. First, the individual decreases in concentration of monomeric alpha-helical forms of the three peptides in an acidified aqueous organic solvent mixture were monitored by electrospray (ES) mass spectrometry. Dipalmitoylated SP-C disappeared with a first-order rate constant of 0.01 h(-1), corresponding to a t(1/2) of 70 hours, while SP-C missing one or two palmitoyl groups disappeared with a rate constant of 0.02 h(-1), t(1/2)=35 hours. This supports the suggestion that the acyl chains stabilise helical SP-C, and that small differences in helix stability can influence fibril formation. The rates of disappearance of the monomeric alpha-helical peptides are much faster than the disappearance of total soluble SP-C (t(1/2)=15 days for SP-C forms soluble after centrifugation at 20,000 g), which suggests that fibril formation is preceded by formation of soluble aggregates. Next, we used matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry to measure hydrogen-->deuterium (H/(2)H) exchange in di-, mono- and non-palmitoylated SP-C in acidified aqueous organic solvents. All three species contain a rigid alpha-helix in their monomeric forms and no difference in deuterium uptake between SP-C with and without palmitoyl groups could be detected. The decreased stability of mono- and non-palmitoylated SP-C observed by ES mass spectrometry is thus not associated with partial unwinding of the helix in solution. Finally, SP-C was shown to unfold during the ES process (where ions are transferred from the solution to the gas phase) and the unfolded forms of di-, mono- and non-palmitoylated SP-C undergo H/(2)H exchange. This, together with the findings from MALDI H/(2)H experiments that the alpha-helix does not exchange, indicates that no partly helical intermediates exist and that the unfolding is highly cooperative.

Amyloid Neuropathies↗
PubMed2001

Electrospray and tandem mass spectrometry in biochemistry.

Over the last 20 years, biological MS has changed out of all recognition. This is primarily due to the development in the 1980s of 'soft ionization' methods that permit the ionization and vaporization of large, polar, and thermally labile biomolecules. These developments in ionization mode have driven the design and manufacture of smaller and cheaper mass analysers, making the mass spectrometer a routine instrument in the biochemistry laboratory today. In the present review the revolutionary 'soft ionization' methods will be discussed with particular reference to electrospray. The mass analysis of ions will be described, and the concept of tandem MS introduced. Where appropriate, examples of the application of MS in biochemistry will be provided. Although the present review will concentrate on the MS of peptides/proteins and lipids, all classes of biomolecules can be analysed, and much excellent work has been done in the fields of carbohydrate and nucleic acid biochemistry.

Bile Acids and Salts↗
PubMed2001

Peptide repertoire of human cerebrospinal fluid: novel proteolytic fragments of neuroendocrine proteins.

Polypeptides in human cerebrospinal fluid (CSF), isolated by phase separation in chloroform-methanol-water and reversed-phase HPLC, were characterised by sequence analysis and mass spectrometry. This identified the presence of peptide fragments of testican, neuroendocrine specific protein VGF, neuroendocrine protein 7B2, chromogranin B/secretogranin I, chromogranin A, osteopontin, IGF-II E-peptide and proenkephalin. The majority of these fragments were generated by proteolysis at dibasic sites, suggesting that they are derived by activities related to prohormone convertase(s). Several of the fragments have previously not been detected, and their functions in CSF or elsewhere are unknown. A characteristic feature of all these fragments is a very high content of acidic residues, in particular glutamic acid. In addition to the fragments of neuroendocrine proteins, endothelin-binding receptor-like protein 2, ribonuclease 1, IGF-binding protein 6, albumin, alpha1-acid glycoprotein 1, prostaglandin-H2 D-isomerase, apolipoprotein A1, transthyretin, beta2-microglobulin, ubiquitin, fibrinopeptide A, and C4A anaphylatoxin were found.

Chromatography, Gel↗
PubMed2001

Gas phase conformation can have an influence on peptide fragmentation.

In the post-genomic era, mass spectrometry is destined to fulfil a central role in biomedical research, and it is in the area of protein identification that mass spectrometry is now most rapidly expanding. An important identification method is to subject a protein to proteolysis and determine the resulting peptide masses and/or primary structure. From such determinations proteins can be identified. Tandem mass spectrometry (MS/MS) is used to determine primary structure and, for high-throughput identification, computer-based automated strategies are a prerequisite. Computer programs are available for such identifications, where simulated MS/MS spectra of amino acid sequences within a database are generated and compared to experimental spectra. Such algorithms take into account empirical rules for peptide fragmentation, rather than specific gas-phase ion chemistry. For example, fragmentation of each peptide bond is usually considered to be equally facile. In reality, this is not the case. Gas-phase ion chemistry bears an important role in determining the abundance of fragment ions in MS/MS spectra. In this communication, the gas-phase ion chemistry responsible for the facile cleavage between Gln and Gly residues is investigated, particularly in relation to Proline Rich Protein-1.

Amino Acid Sequence↗
PubMed2001

Gln-Gly cleavage: a dominant dissociation site in the fragmentation of protonated peptides.

An understanding of the gas-phase dissociation of protonated peptides within the mass spectrometer is essential for automated high-throughput protein identification. In this communication we describe a facile cleavage of the Gln-Gly peptide bond under low-collisional energy conditions. A variety of synthetic peptides have been analysed where key amino acids have been substituted within the sequence PQGPPQQGGR, which is a consensus repeat present in the tryptic peptides of acidic proline-rich protein 1 (PRP-1). The collision-induced dissociation spectra obtained from the PRP-1 tryptic peptides and the synthetic peptides indicate that facile Gln-Gly cleavage occurs when an X-Gln-Gly-Y sequence is present in a peptide, where X is any amino acid and Y any amino acid other than Gly.

Amino Acid Sequence↗
PubMed2001

Gln-Gly cleavage: correlation between collision-induced dissociation and biological degradation.

Tryptic digestion of the 150-residue human acidic salivary proline-rich protein 1 (PRP-1) generated eight peptides, two of which corresponded to the N-terminal 30-residue segment. In each of the other six tryptic peptides, a consensus repeat with the structure PQGPPQQGG was present. A facile Gln-Gly cleavage between the second and the third residues of the repeat was observed during collision-induced dissociation experiments. We postulate possible mechanisms to account for this reactivity, involving attack on the peptidyl carbonyl group by the Gln sidechain. Significantly, the Gln-Gly cleavage has been shown to be biologically important in the bacterial degradation of PRPs in saliva, generating bacteria-binding Pro-Gln C-termini. We suggest a link between the gas-phase chemistry and the biochemical degradation of these molecules.

Amino Acid Sequence↗
PubMed2001

Intestinal iron uptake determined by divalent metal transporter is enhanced in HFE-deficient mice with hemochromatosis.

BACKGROUND & AIMS: Overexpression of duodenal divalent metal transporter (DMT1) messenger RNA occurs in hemochromatosis and HFE-knockout mice, suggesting that DMT1 mediates enhanced absorption of iron; however, increased expression of functional DMT1 protein has yet to be substantiated. We examined the role of DMT1 and the mucosal iron uptake defect in HFE-knockout mice. METHODS: Unidirectional iron uptake of 59Fe by small intestinal mucosa in vitro was compared between matched pairs of HFE-knockout and wild-type mice. DMT1-specific antibodies were used to block iron transport and to quantify duodenal protein expression. RESULTS: Ferrous iron uptake at 3.5-450 micromol/L was greatly enhanced in HFE-knockouts compared with wild-type, the apparent V(max) for Fe2+ transport being doubled (P < 0.01). Supplied as Fe3+, uptake was only enhanced in HFE-knockouts at < or =18 micromol/L, when the iron was almost completely converted to Fe2+ by mucosal ferrireductases. DMT1 antibody reduced the apparent Vmax for mucosal Fe2+ transport in HFE-knockouts to below wild-type control values (P < 0.02); immunoreactive mucosal DMT1 protein was increased nearly 2-fold in HFE-knockouts (P < 0.01). CONCLUSIONS: Disruption of the HFE gene up-regulates functional DMT1 transporters and enhances uptake of ferrous iron by this mechanism; DMT1 also mediates increased uptake after reduction of ferric iron presented at physiological concentrations.

Animals↗
PubMed2001

Membrane activity of (Cys48Ser) lung surfactant protein B increases with dimerisation.

One of the possible functions of lung surfactant protein B (SP-B), an hydrophobic membrane-associated saposin-like protein, is to reduce the alveolar surface tension by promoting insertion of phospholipids into the air/liquid interface of the lung. SP-B is a covalent homodimer; Cys48 of two polypeptides form an intermolecular disulphide bond. In order to test whether dimerisation of SP-B is important for surfactant function, transgenic mice which express (Cys48Ser) human SP-B in a mouse SP-B null background were generated. In previous studies (Cys48Ser)SP-B showed a concentration-dependent in vitro activity, suggesting that it may form non-covalent dimers. Here (Cys48Ser)SP-B isolated from bronchoalveolar lavage of transgenic mice was studied at different concentrations by circular dichroism (CD) spectroscopy, pulsating bubble surfactometry, mass spectrometry and reversed-phase HPLC. The results indicate that (Cys48Ser)SP-B, both in a phospholipid environment and in organic solvents, is largely monomeric and exhibits low activity at concentrations lower than 1 -2 microM, while at higher concentrations it forms non-covalent dimers, which are nearly functionally equivalent to native SP-B in vitro. Furthermore, electrospray mass spectrometry showed that more dimers were found relative to the monomer when the polarity of the solvent was decreased, and when the concentration of SP-B increased. (Cys48Ser)SP-B also eluted earlier than native SP-B in reversed-phase HPLC. Taken together, these results indicate that a polar surface is buried upon dimerisation, thereby promoting formation of interchain ion pairs between Glu51-Arg52' and Glu51'-Arg52.

Amino Acid Substitution↗
PubMed2001

Identification of unusual 7-oxygenated bile acid sulfates in a patient with Niemann-Pick disease, type C.

Niemann-Pick disease, type C, was diagnosed in a 3-month-old boy with hepatosplenomegaly, mild signs of cholestasis, hepatic inflammation and extramedullary erythropoiesis, together with chronic airway disease. He developed muscular hypotonia, psychomotor retardation, rickets, and signs of peripheral neuropathy. The patient was found to excrete abnormal amounts of unusual bile acids in urine at 3 and 5 months of age. These acids were shown to have a 3beta-hydroxy-Delta(5) structure and to carry an oxo or hydroxy group at C-7. They were sulfated at C-3 and nonamidated or conjugated with glycine or taurine at C-24. Part of the 7-hydroxy acids, presumably the 7beta-hydroxylated one, was also conjugated with N-acetylhexosamine, probably N-acetylglucosamine, at the 7-hydroxy group. Possible metabolic pathways for the formation of the 7-oxo and 7beta-hydroxycholenoic acids are discussed. Based on previous data concerning the effects of 3beta-hydroxy-Delta(5) bile acids on bile acid transport, it is suggested that the formation of such bile acids is responsible for the cholestasis in this patient.

Bile Acids and Salts↗
PubMed2000

Ring opening of benzo[a]pyrene in the germ-free rat is a novel pathway for formation of potentially genotoxic metabolites.

The metabolism of benzo[a]pyrene (BP) is known to lead to a large number of oxygenated compounds, some of which can bind covalently to DNA. We have studied the integrated metabolism of BP in vivo in germ-free rats given (14)C-labeled BP. Urinary metabolites were separated into groups according to acidity using lipophilic ion exchangers. The groups were analyzed by mass spectrometry and were further fractionated by high-performance liquid chromatography. The fraction of urinary metabolites previously shown to contain N-acetylcysteine and glucuronic acid conjugates was found to contain derivatives of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid as major components. These compounds, which were identified by mass spectrometry and NMR, accounted for about 30% of the total metabolites in urine, demonstrating that, surprisingly, ring opening is a major pathway for metabolism of BP in the germ-free rat. The dicarboxylic acid may be excreted in urine as an ester glucuronide. By using the single cell gel electrophoresis or COMET assay, we were able to demonstrate that the anhydride of 7-oxo-benz[d]anthracene-3, 4-dicarboxylic acid was an efficient inducer of DNA damage. Taken together, these results indicate that the novel ring opening metabolic pathway may provide alternative mechanisms for the toxicity of BP.

Animals↗
PubMed2000

A novel Ser O-glucuronidation in acidic proline-rich proteins identified by tandem mass spectrometry.

Human acidic proline-rich salivary protein PRP-1 and its C-terminally truncated form PRP-3 were analyzed by electrospray tandem mass spectrometry. Post-translational modifications were detected and characterized. A pyroglutamic acid residue was demonstrated at the N-terminus, Ser-8 and Ser-22 were shown to be phosphorylated and an O-linked glucuronic acid conjugation was identified. The latter modification was located to Ser-17 and found to be present in approximately 40% of the polypeptides.

Amino Acid Sequence↗
PubMed2000

Analysis of oxosteroids by nano-electrospray mass spectrometry of their oximes.

A method for the analysis of neutral oxosteroids by electrospray mass spectrometry is described. The oxosteroids are converted into their oximes by treatment with hydroxyammonium chloride in aqueous methanol. Intense peaks corresponding to protonated oxime molecules are observed in nano-electrospray mass spectra. The detection limits for the oximes of progesterone, pregnenolone and dehydroepiandrosterone were 2.5, 5 and 25 pg/microL, respectively, approximately 20 times lower than for the underivatised steroids. The signal intensities were proportional to the concentration of the steroids in the range of 500 to 2.5 pg/microL. Fragmentation by collision-induced dissociation (CID) was studied using oximes of 28 model steroids carrying an oxo group at C-3, C-17 or C-20. Some of the steroid oximes were labelled with deuterium or (15)N. Fragment ions were observed which yielded useful structural information. Upon CID, protonated oximes of 3-oxo-Delta(4)-steroids produced abundant ions by cleavage through the B-ring and by loss of the side chain, while protonated oximes of saturated 3-oxosteroids did not give abundant ions by cleavage through the B-ring. Protonated oximes of 20-oxosteroids unsubstituted at C-21, C-17 or C-16 produced a characteristic ion at m/z 86 containing the side chain, C-16 and C-17. Protonated oximes of steroids containing only a 17-oxo group gave fewer ions of diagnostic value. Coupled with the selective isolation of steroid oximes from a biological matrix this method of derivatisation and CID may be used for the analysis of neutral oxosteroids in biological samples.

Dehydroepiandrosterone↗
PubMed2000

Electrospray tandem mass spectrometry in the rapid identification of alpha-chain haemoglobin variants.

The potential use of electrospray tandem mass spectrometry in the rapid characterisation of haemoglobin variants found in the Swedish population has been assessed. Analysis times of the order of 5 -10 min were routinely achieved, and identification of variants using mass spectrometry as the sole analytical technique was possible. However, additional information, readily available from isoelectric focusing experiments, made identification simpler and more secure. In the present communication we report on the identification of the alpha-chain variants, Hb Russ, Hb Le Lamentin and Hb Q-Iran. The identifications were confirmed by the use of nucleotide sequencing techniques.

Genetic Variation↗