Biomedical subjects

R A Harry

Publications and source records attributed to R A Harry.

4 recordsLinked to original sources

PubMed2001

Separation and identification of rat skeletal muscle proteins using two-dimensional gel electrophoresis and mass spectrometry.

Skeletal muscle plays a major role in whole body protein metabolism, and changes in the rates of synthesis and degradation of proteins are likely to lead to characteristic changes in the amounts of different proteins in muscle under various physiological and pathological conditions. This paper demonstrates the feasibility of a proteomic approach to analyzing the protein composition of skeletal muscle. We report here the initial establishment of two-dimensional gel electrophoresis (2-D PAGE) reference maps for mixed skeletal muscle taken from the abdominal wall of a normal adult rat. We used immobilized pH gradients of 3-10 (non-linear) and 4-7 (linear), and matrix assisted laser desorption/ionization--time of flight mass spectrometry for protein identification by peptide mass fingerprinting. More than 600 protein spots were detected on each gel, of which 100 were excised and characterized. In-gel digestion followed by peptide mass fingerprinting enabled tentative identification of 74 of these, which included a wide range of myofibrillary and sarcoplasmic proteins. This database should provide the nucleus of a valuable resource for investigation of the biochemical basis of skeletal muscle pathologies in general and such specific disorders as alcoholic myopathy and injury.

Animals↗

PubMed2000

Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes.

While the classical silver stain has been the method of choice for high sensitivity protein visualization on two-dimensional gel electrophoresis (2-D PAGE), post-electrophoretic fluorescent staining with the SYPRO group of dyes has emerged to challenge silver staining for proteome analysis. The latter offers improved sensitivity, higher dynamic range and easy handling. However, most of the published data were derived from analysis of 1-D gel separations. In this work, we have focused on three commercially available fluorescent dyes, SYPRO Ruby, SYPRO Orange and SYPRO Red (Molecular Probes, Eugene, OR, USA) and studied their sensitivity and dynamic range on 2-D PAGE. The use of a multiwavelength fluorescent scanner to image 2-D protein profiles visualized with fluorescent staining is discussed, and a detailed comparison with analysis by silver staining is also provided. These results demonstrate the advantages of using SYPRO dyes, which are in agreement with the literature based on 1-D gel electrophoresis, and give a more realistic understanding of the performance of these fluorescent dyes with 2-D PAGE.

Dextrans↗

PubMed2000

A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry.

The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.

Animals↗

PubMed2000

A prospective series of out-patient endoscopic retrograde cholangiopancreatography.

OBJECTIVE: Out-patient endoscopic retrograde cholangiopancreatography (ERCP) is widely practised but the safety of this approach has only been addressed in a limited number of prospective series mainly from specialist North American centres. Our objective was to determine prospectively the safety and admission rates of out-patient ERCPs. STUDY DESIGN AND PARTICIPANTS: Patients were selected for out-patient ERCP if in relatively good health, without major risk factors for complications following ERCP and with adequate social support. Our series consisted of 136 consecutive cases of which 82 were therapeutic. SETTING AND OUTCOME MEASURES: A district general hospital in the UK, which also performs ERCPs for neighbouring health districts. Out-patient ERCP patients were followed up at 30 days using standard criteria for defining complications. RESULTS: Procedures were 60 biliary sphincterotomy, 10 stone removal, nine stenting procedures, two dilatations and one pancreatic intervention. Complications were pancreatitis in seven patients (six moderate severity, one mild), cholangitis in three patients, haemorrhage in one patient. Nine patients required admission for complications, two from the endoscopy unit and seven from home; their average in-patient stay was 6 days. Seventeen patients were admitted for observation or for further management. There was one death unrelated to ERCP. Overall, 110 of 136 patients did not require inpatient care following out-patient ERCP. CONCLUSIONS: Our complication rates were similar to those of other series. Out-patient ERCP for selected cases, with adequate post-discharge arrangements for advice and readmission, appears safe and would reduce healthcare costs.

Adult↗