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.
Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS-CoV) isolated from Vero E6 cells infected with the BJ-01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS-CoV in the cultured supernatant of virus infected-Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in-gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS-CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix-assisted laser desorption/ionization-mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N-terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS-PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS-CoV was cleaved in vitro by caspase-3 and -6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase-3 rather than that of caspase-6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.
Recombinant human erythropoietin (rhEPO) has been extensively used as pharmaceutical product for treatment of anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics and in vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for its quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple enzyme digestion, hydrophilic interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high resolution and high accuracy (HR/AM) mass spectrometry on an Orbitrap. Totally, 74 intact glycopeptides from four glycosylation sites at N24, N38, N83 and O126 were identified with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR/AM data allowed relative quantification of glycoforms. Our results could be extended to the quality control of rhEPO or help to establish detection approaches for glycosylation of other proteins.
Cardiovascular diseases (CVD) are the leading cause of death in the world. However, due to the limited effectiveness and potential adverse effects of current treatments, the long-term prognosis of CVD patients is still discouraging. In recent years, several studies have found that berberine (BBR) has broad application prospects in the prevention and treatment of CVD. Due to its effectiveness and safety for gastroenteritis and diarrhea caused by bacterial infections, BBR has been widely used in China and other Asian countries since the middle of the last century. The development of pharmacology also provides evidence for the multi-targets of BBR in treating CVD. Researches on CVD, such as arrhythmia, atherosclerosis, dyslipidemia, hypertension, ischemic heart disease, myocarditis and cardiomyopathy, heart failure, etc., revealed the cardiovascular protective mechanisms of BBR. This review systematically summarizes the pharmacological research progress of BBR in the treatment of CVD in recent years, confirming that BBR is a promising therapeutic option for CVD.
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