Abstract: The full-length dystrophin protein isoform of 427 kDa (Dp427), the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normal versus dystrophic mdx-4cv skeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that the mdx-4cv mouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen in mdx-4cv muscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context.
Abstract: We produced a tear proteome of the genome mouse, C57BL/6, that contained 139 different protein identifications: 110 from a two-dimensional (2D) gel with subsequent trypsin digestion, 19 from a one-dimensional (1D) gel with subsequent trypsin digestion and ten from a 1D gel with subsequent Asp-N digestion. We compared this tear proteome with a C57BL/6 mouse saliva proteome produced previously. Sixteen of the 139 tear proteins are shared between the two proteomes, including six proteins that combat microbial growth. Among the 123 other tear proteins, were members of four large protein families that have no counterparts in humans: Androgen-binding proteins (ABPs) with different members expressed in the two proteomes, Exocrine secreted peptides (ESPs) expressed exclusively in the tear proteome, major urinary proteins (MUPs) expressed in one or both proteomes and the mouse-specific Kallikreins (subfamily b KLKs) expressed exclusively in the saliva proteome. All four families have members with suggested roles in mouse communication, which may influence some aspect of reproductive behavior. We discuss this in the context of functional adaptation involving tear and saliva proteins in the secretions of mouse lacrimal and salivary glands, respectively.
Abstract: Developing a urine test to detect bladder tumours with high sensitivity and specificity is a key goal in bladder cancer research. We hypothesised that bladder cancer-specific glycoproteins might fulfill this role. Lectin-ELISAs were used to study the binding of 25 lectins to 10 bladder cell lines and serum and urine from bladder cancer patients and non-cancer controls. Selected lectins were then used to enrich glycoproteins from the urine of bladder cancer patients and control subjects for analysis by shotgun proteomics. None of the lectins showed a strong preference for bladder cancer cell lines over normal urothlelial cell lines or for urinary glycans from bladder cancer patients over those from non-cancer controls. However, several lectins showed a strong preference for bladder cell line glycans over serum glycans and are potentially useful for enriching glycoproteins originating from the urothelium in urine. Aleuria alantia lectin affinity chromatography and shotgun proteomics identified mucin-1 and golgi apparatus protein 1 as proteins warranting further investigation as urinary biomarkers for low-grade bladder cancer. Glycosylation changes in bladder cancer are not reliably detected by measuring lectin binding to unfractionated proteomes, but it is possible that more specific reagents and/or a focus on individual proteins may produce clinically useful biomarkers.
Abstract: Damage to normal lung tissue is a limiting factor when ionizing radiation is used in clinical applications. In addition, radiation pneumonitis and fibrosis are a major cause of mortality following accidental radiation exposure in humans. Although clinical symptoms may not develop for months after radiation exposure, immediate events induced by radiation are believed to generate molecular and cellular cascades that proceed during a clinical latent period. Oxidative damage to DNA is considered a primary cause of radiation injury to cells. DNA can be repaired by highly efficient mechanisms while repair of oxidized proteins is limited. Oxidized proteins are often destined for degradation. We examined protein oxidation following 17 Gy (0.6 Gy/min) thoracic X-irradiation in C57BL/6J mice. Seventeen Gy thoracic irradiation resulted in 100% mortality of mice within 127–189 days postirradiation. Necropsy findings indicated that pneumonitis and pulmonary fibrosis were the leading cause of mortality. We investigated the oxidation of lung proteins at 24 h postirradiation following 17 Gy thoracic irradiation using 2-D gel electrophoresis and OxyBlot for the detection of protein carbonylation. Seven carbonylated proteins were identified using mass spectrometry: serum albumin, selenium binding protein-1, alpha antitrypsin, cytoplasmic actin-1, carbonic anhydrase-2, peroxiredoxin-6, and apolipoprotein A1. The carbonylation status of carbonic anhydrase-2, selenium binding protein, and peroxiredoxin-6 was higher in control lung tissue. Apolipoprotein A1 and serum albumin carbonylation were increased following X-irradiation, as confirmed by OxyBlot immunoprecipitation and Western blotting. Our findings indicate that the profile of specific protein oxidation in the lung is altered following radiation exposure.
Abstract: Dry edible bean (Phaseolus vulgaris L.) is a globally relevant food crop. The bean genome was recently sequenced and annotated allowing for proteomics investigations aimed at characterization of leaf phenotypes important to agriculture. The objective of this study was to utilize a shotgun proteomics approach to characterize the leaf proteome and to identify protein abundance differences between two bean lines with known variation in their physiological resistance to biotic stresses. Overall, 640 proteins were confidently identified. Among these are proteins known to be involved in a variety of molecular functions including oxidoreductase activity, binding peroxidase activity, and hydrolase activity. Twenty nine proteins were found to significantly vary in abundance (p-value < 0.05) between the two bean lines, including proteins associated with biotic stress. To our knowledge, this work represents the first large scale shotgun proteomic analysis of beans and our results lay the groundwork for future studies designed to investigate the molecular mechanisms involved in pathogen resistance.
Abstract: Lotus (Nelumbo nucifera Gaertn.) seed proteome has been the focus of our studies, and we have recently established the first proteome dataset for its mature seed endosperm. The current study unravels the immature endosperm, as well as the embryo proteome, to provide a comprehensive dataset of the lotus seed proteins and a comparison between the mature and immature endosperm tissues across the seed’s development. One-dimensional gel electrophoresis (SDS-PAGE) linked with tandem mass spectrometry provided a protein inventory of the immature endosperm (122 non-redundant proteins) and embryo (141 non-redundant proteins) tissues. Comparing with the previous mature endosperm dataset (66 non-redundant proteins), a total of 206 non-redundant proteins were identified across all three tissues of the lotus seed. Results revealed some significant differences in proteome composition between the three lotus seed tissues, most notably between the mature endosperm and its immature developmental stage shifting the proteins from nutrient production to nutrient storage.