Colin H. Wheeler

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.

Bovine dilated cardiomyopathy : proteomic analysis of an animal model of human dilated cardiomyopathy

Bovine hereditary dilated cardiomyopathy (bCMP) is endemic in Switzerland and hearts from diseased animals display important clinical and biochemical similarities to human DCM. Recent research has identified at least one protein (myoglobin) to be significantly reduced in bovine DCM. Using a proteomic approach, we have separated over 1125 protein species from bovine ventricular tissue. Gel analysis and protein characterisation have identified a number of proteins whose abundance is significantly altered in bovine DCM. Twenty‐four proteins are of decreased abundance in diseased tissue, whilst 11 proteins are of increased abundance in the diseased state. A combination of amino acid compositional analysis, peptide mass profiling, N‐terminal microsequencing and MultiIdent (http://www.expasy.ch/sprot/multiident.html) has been employed in order to elucidate the identities of the differentially expressed proteins. Using these techniques we have currently determined the identity of 12 of the 35 altered proteins. We have also detected three proteins that are differentially expressed in genotypically diseased but phenotypically normal animals, identifying a possible mechanism for the onset of the disease. The possibility that inappropriate ubiquination of proteins plays an important role in the disease is discussed. A database of bovine proteins is currently being established. The identity of the proteins affected, together with a comparison of the human and bovine expression patterns, is displayed.

Characterization and identification of latex allergens by two-dimensional electrophoresis and protein microsequencing ☆ ☆☆ ★

BACKGROUND Proteins of natural rubber latex cause IgE-mediated sensitization in 3% to 18% of health care workers and in up to 50% of patients with spina bifida. OBJECTIVE This study was aimed at the generation of a comprehensive latex protein database by two-dimensional electrophoresis (2-DE). METHODS Proteins extracted from fresh Hevea brasiliensis latex were separated by 2-DE. IgE-reactive proteins were analyzed by immunoblotting with sera of health care workers with latex allergy. Protein microsequencing and monoclonal antibodies were used to identify the latex allergens. RESULTS The latex C-serum 2-DE map was very complex and exhibited about 200 distinct polypeptides. The proteins eluted from the latex particles consisted primarily of two groups of acidic proteins located in the 8 to 14 kd and 22 to 24 kd areas of the 2-DE map. Major IgE-reactivity was detected with C-serum proteins in the 56, 45, 30, 20, 14, and <6.5 kd areas of the immunoblots. The 8 to 14 kd particle proteins exhibited distinct IgE reactivity, whereas the 22 to 24 kd proteins were not stained. Seven of the soluble IgE-reactive protein spots showed high homology with enolase, superoxide dismutase, triosephosphate isomerase, proteasome subunit, and chitinase and represent previously undescribed latex allergens; whereas nine protein spots corresponded to known latex allergens, namely prohevein, hevein, prohevein C-domain, and hevamine. As identified by monoclonal antibodies, the IgE-reactive latex particle proteins mainly represent the allergenic rubber elongation factor. CONCLUSIONS Two-dimensional electrophoresis, followed by immunoblotting and protein microsequencing, can rapidly identify a large number of IgE-binding latex proteins. The 2-DE latex maps generated will provide valuable information for the development of strategies to isolate the relevant latex allergens. Because the novel latex allergens are common plant enzymes, they may also act as cross-reacting proteins in various foods.

Changes in myocardial protein expression in pacing-induced canine heart failure

Canine rapid ventricular pacing produces a low output cardiomyopathic state which is similar to dilated cardiomyopathy. In this study dogs were paced at 245 beats per minute (bpm) for 3—4 weeks until signs of heart failure were apparent. Unpaced dogs were used as controls. A previous study identified myocardial protein changes in the pH region 4—7 following ventricular pacing by using two‐dimensional electrophoresis (2‐DE) (Heinke et al., Electrophoresis 1998 19, 2021—2030). Many of these proteins were associated with mitochondria, energy metabolism within the cardiomyocyte, the cytoskeleton and calcium cycling. The present study aimed to examine the proteins migrating in the more basic region of the 2‐DE pattern using immobilised pH gradient 3—10 strips to separate myocardial proteins. The expression of 31 proteins was altered in the paced myocardium: 21 were decreased and 10 increased. Following the identification of 23 of these spots by either amino acid compositional analysis or peptide mass fingerprinting or a combination of both, we confirm that many of the proteins whose expression is altered following ventricular pacing are associated with the mitochondria and energy production within the cardiomyocyte, including creatine kinase M, triosephosphate isomerase, phosphoglycerate mutase, cytochrome c oxidase, cytochrome b5, hydroxymethyl glutaryl CoA synthase, myoglobin, and 3,2‐trans‐enoyl‐CoA transferase. Additionally, the cytoskeletal protein actin was increased in the paced hearts. These results strongly support the notion that energy production is impaired and mitochondrial dysfunction is involved in the development of heart failure in the paced dog.

Identification, distribution and physiological activity of three novel neuropeptides of Lymnaea: EFLRlamide and pQFYRlamide encoded by the FMRFamide gene, and a related peptide.

We are interested in analysing the detailed modulation of defined neuronal systems by multiple neuropeptides encoded in the FMRFamide locus of the snail Lymnaea. Cloning of the FMRFamide gene has predicted the existence of two novel peptides previously unknown from biochemical analysis, the pentapeptides EFLRlamideand QFYRlamide. These peptides may form part of a new family of peptides sharing the sequence motif –FXRlamide. In this paper we adopt a novel approach to first identify and characterize –FXRlamide‐like peptides in extracts from the central nervous system of Lymnaea. By a combination of high‐performance liquid chromatography (HPLC) and continuous‐flow fast atom bombardment mass spectrometry, we identify three novel peptides: EFLRlamide, pQFYRlamide and pQFLRlamide. The first two are those predicted in exon II of the FMRFamide locus whereas the last is, interestingly, a product which cannot be derived from post‐translational modification of the predicted peptides but must be encoded by as yet unidentified nucleotide sequences. A specific antibody raised to EFLRlamide, and immuno reactive to all three peptides, revealed EFLRlamide‐like expression throughout the central nervous system in the same cells where exon II is transcribed and the peptide SEEPLY (a post‐translational product of exon II) was localized. Additional cells, however, were also identified. Immunoreactivity was mapped in a number of identified neurons in the central nervous system, including two heart cardio excitatory motoneurons, the Ehe cells (E heart excitors of the visceral ganglion) and penialmotoneurons in the right cerebral ganglion. The peripheral tissues (heart and penial complex) that the serespective classes of neurons innervate also exhibited EFLRlamide immunoreactivity. The central and peripheral localization of EFLRlamide‐like immunoreactivity suggested that EFLRlamide/pQFYRlamide may have an important physiological role in both these peripheral systems as well as in the central nervous system. This was confirmed by physiological experiments that showed that EFLRlamide and pQFYRlamide inhibited many centralneurons and in particular the Bgp neurons in the right parietal ganglion. EFLRlamide had complex biphasic effects on the frequency of heart‐beat: an initial inhibitory response was followed by a long‐lasting increase in the rate of beating. Taken together with earlier work, this study now completes the analysis and localization of the full set of post‐translational products of the FMRFamide precursor in Lymnaea and supplies further evidence towards the characterization of the physiological systems which such peptides may modulate in concert.

Transplant Associated Coronary Artery Disease

Heart transplantation is the clinically acceptable treatment for end-stage heart failure. Since the development of better immunosuppressive regimes and particularly the introduction of Cyclosporin A in 1983, which combats cellular rejection, short-term survival for heart transplant recipients has increased steadily with survival at 1 year being in the order of 85% (Kriett and Kaye 1990). The major medium to long-term complication is development of a proliferative occlusive disease of the vasculature of the allograft. This disease, variously described as transplant associated coronary artery disease (TxCAD; Rose and Dunn 1993), cardiac allograft vasculopathy (CAV; Hosenpud et al. 1992), accelerated coronary artery sclerosis (ACS; Yacoub and Rose, 1994) and graft coronary artery sclerosis (GCA; Schoen and Libby 1991), is a rapidly progressing disease which causes blockage of the coronary arteries. The incidence of TxCAD varies between heart transplant centres. Harefield Hospital, U.K. has an incidence of 6% at 1 year increasing to 17% at 3 years (see Dunn and Rose, 1993), however, other centres have reported incidences as high as 10%–20% at 1 year, 25%–40% at 3 years and at least 40%–50% at 5 years (Gao et al. 1989; Uretsky et al. 1987).

Abstract 5071: Application of a novel protein biomarker discovery platform to identify biomarkers for improved diagnosis of prostate cancer

The issues surrounding the use of prostate-specific antigen (PSA) in the diagnosis of prostate cancer (PCa) are well documented and the need for a molecular diagnostic test with greater discriminatory power is clear. The development of autoantibodies associated with prostate cancer has also been described. In general, the appearance of such antibodies can precede disease symptoms by many years, making them attractive as potential biomarkers for early diagnosis. We have developed a unique “functional protein” array platform which utilises correctly folded proteins and has the ability to display native, discontinuous epitopes. The reproducibility of the platform is exceptionally good, making it possible to screen statistically meaningful numbers of samples. Following on from a successful pilot study, where panels of autoantibody biomarkers exhibiting a specificity and sensitivity for PCa superior to PSA were identified using the platform 1 , we have validated this approach in a large-scale analytical study. The current analytical study involving approximately 1800 samples was primarily designed to identify panels of biomarkers with the ability to distinguish between PCa (n=400) and control samples from patients with benign prostatic hypertrophy (BPH, n=406). BPH can present with similar symptoms to PCa and can also result in elevated PSA levels. Additional sample cohorts included prostatitis, other cancers of various origin and non-disease/healthy controls (n=400). All samples were age, ethnicity and gender matched. The products of 1296 unique genes (1330 proteins), chosen for their association with disease, signal transduction and cancer autoimmunity, are immobilized on each array. Serum samples (n=1781) were analysed using the arrays as described previously 1 . Data were split into test and training sets and analyzed with several classification algorithms to identify classifiers which would successfully distinguish case from control samples. Data were repeatedly split into test and training sets and analysis cycles repeated until a stable set of classifiers was identified. At the time of writing, data is still under analysis; however, early indications are encouraging and suggest that panels of biomarkers with a performance that significantly exceeds that of PSA may be identified. Full results of the study will be presented. 1 McAndrew et al Development of a panel of biomarkers for the diagnosis of prostate cancer (2010) Molecular Diagnostics in Cancer Therapeutic Development conference Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5071. doi:10.1158/1538-7445.AM2011-5071