We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.
Atrial fibrillation (AF) is a common arrhythmia that increases the risk of stroke and heart failure. Here, we have shown that mast cells, key mediators of allergic and immune responses, are critically involved in AF pathogenesis in stressed mouse hearts. Pressure overload induced mast cell infiltration and fibrosis in the atrium and enhanced AF susceptibility following atrial burst stimulation. Both atrial fibrosis and AF inducibility were attenuated by stabilization of mast cells with cromolyn and by BM reconstitution from mast celldeficient WBB6F1-Kit W/W-v mice. When cocultured with cardiac myocytes or fibroblasts, BM-derived mouse mast cells increased platelet-derived growth factor A (PDGF-A) synthesis and promoted cell proliferation and collagen expression in cardiac fibroblasts. These changes were abolished by treatment with a neutralizing antibody specific for PDGF α-receptor (PDGFR-α). Consistent with these data, upregulation of atrial Pdgfa expression in pressure-overloaded hearts was suppressed by BM reconstitution from WBB6F1-Kit W/W-v mice. Furthermore, injection of the neutralizing PDGFR-α-specific antibody attenuated atrial fibrosis and AF inducibility in pressure-overloaded hearts, whereas administration of homodimer of PDGF-A (PDGF-AA) promoted atrial fibrosis and enhanced AF susceptibility in normal hearts. Our results suggest a crucial role for mast cells in AF and highlight a potential application of controlling the mast cell/PDGF-A axis to achieve upstream prevention of AF in stressed hearts.
To identify proteins that could be molecular targets for diagnosis and treatment of hepatitis C virus-related hepatocellular carcinoma (HCV-related HCC), we used a proteomic approach to analyze protein expression in samples of human liver. Twenty-six pairs of tumorous and corresponding nontumorous liver samples from patients with HCV-related HCC and six normal liver samples were analyzed by two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. One of the numerous spots that showed stronger intensity in tumorous than in nontumorous samples was identified as alpha enolase, a key enzyme in the glycolytic pathway. Expression of this protein increased with tumor dedifferentiation and was significantly higher in poorly differentiated HCC than in well-differentiated HCC. This pattern was reproduced by immunoblot analysis and immunohistochemistry. Expression of alpha enolase also correlated positively with tumor size and venous invasion. These results suggest that alpha enolase is one of the candidates for biomarkers for tumor progression that deserves further investigation in HCV-related HCC.
The prognosis of patients with pancreatic cancer is very poor because of late diagnosis and the lack of response to various therapies. We tried to identify proteins that might be available for early diagnosis and effective therapies by proteomic profiling of pancreatic cancer tissues. Pancreatic cancerous and paired non-cancerous tissues obtained from surgical resections or autopsies of 10 patients were analyzed by two-dimensional gel electrophoresis. The differential display showed 11 spots whose expression was increased in cancerous tissues compared with the paired non-cancerous tissues. The liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) system identified the spots as ·enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triosephosphate isomerase, transgelin, calmodulin, superoxide dismutase(Mn) mitochondrial precursor, glutathione S-transferase P, cyclophilin A, protein disulfide isomerase A3 precursor, and apolipoprotein A-I precursor. Two of the 11 spots were detected as GAPDH. We noticed that 4 of 11 spots were enzymes involved in glycolytic pathway. Increased glycolysis in cancer cells has been regarded as the effect of intratumoral hypoxia and is possibly associated with tumor invasion, metastasis or resistance to therapies. These glycolytic proteins and transgelin, were confirmed by Western blotting and immunohistochemistry.Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TPI, triosephosphate isomerase; 2-DE, two-dimensional gel electrophoresis; MnSOD, superoxide dismutase(Mn) mitochondrial precursor; PDI-A3, protein disulfide isomerase A3 precursor; GST-p, glutathione S-transferase P
Pancreatic cancer remains a devastating disease and >96% of patients with pancreatic cancer do not survive for more than 5 years. Gemcitabine (2'-deoxy-2'-difluorodeoxycytidine: Gemzar) appears to be the only clinically effective drug for pancreatic cancer, but it has little impact on outcome. Proteomic analysis of gemcitabine-sensitive cells (KLM1) and resistant pancreatic cells (KLM1-R) was performed to identify target proteins of the gemcitabine. We found seven proteins, HSP27, peroxiredoxin 2, endoplasmic reticulum protein ERp29 precursor, 6-phosphogluconolactonase, triosphospate isomerase, α enolase, and nucleophosmine that could play a role in determining the sensitivity of pancreatic cancer to gemcitabine. We knocked down HSP27 in KLM1-R and the sensitivity to gemcitabine was restored. In addition, increased HSP27 expression in tumor specimens was related to higher resistibility to gemcitabine in patients of pancreatic cancer. HSP27 may play an important role in the resistibility to gemcitabine, and it could also be a possible biomarker for predicting the response of pancreatic cancer patients to treatment with gemcitabine.
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