| Plenty of modifications were found to exist in histones, a protein family wrapped with DNA. These post-translational modifications play an important role in regulating gene expression. In analyzing the open modification search results of human histones from tumor and adjacent tissues in kidney cancer patients, different residues were identified with +42 modification, they were lysine, serine, threonine, alanine and glutamic acid. Restricted modification search confirmed lysine and two novel sites of +42modification-serine and threonine, it seemed that the +42 modification trended to be acetylation. Acetylation is one of the general post-transcriptional modifications in histones, well-known locus of acetylation includes residues at amino terminal of proteins and lysine sites. This research showed that, excepted for lysine, many serine and threonine non-amino-terminal sites were identified with +42 modification. Site specific antibody further proved the real existence of acetylation on a conserbative site-threonine 22 in histone H3. Thus, acetylation of serine and threonine in histones was confirmed, and these new acetylation sites were identified in a certain number of human cell histones.BackgroundsMass spectrometry analysis has become an important way in proteomics, with the raising of mass accuracy and the optimization of analysis methods, qualitative mass spectrometry can not meet the requirement, increasing number of quantitative methods has been used in basic and clinical medical study. Nowadays, quantification with labels has been applied to clinical research, while it can hardly be exensively used for its expensive cost and complicate operation. Thus, the label-free quantification method is more suitable for massive preliminary study.AimsUsing a label-free method based on primary spectra, comparing the primary mass spectra from ethanol induced hepatocellular carcinoma cell line HepG2, make a relative quantitation of HepG2 total proteins, thus explore the mechanism of alcoholic liver disease.Methods1. Cell processing. HepG2 was treated with ethanol, the total protein in cell lysate was underwent in-solution-digestion.2. Mass spectrometric analysis. The total digested peptides was analyzed by liquid chromatography-two stage mass spectrometry (LC-MS/MS).3. Quantitation. The primary spectra from ethanol induced and control cell lines were compared by Progenesis LC-MS, a commercial quantification software. Quantitative value is the integral evaluation of every peptide peak. The difference of proteins is counted with the quantitation of each peptide.4. Bioinformatics analysis. Proteins with difference in amount counted by Progenesis were further analyzed by several open protein databases, such as Panther and String, to explore the assortment and intercommunication among these proteins.Results1. The ms/ms data were search by Mascot software. The number of identified protein was 1188 and 1798 in ethanol induced and control cells, the total peptides was 18564 and 19505, and the number of total ms/ms spectra was 71089 and 55203, respectively.2.25462 ion peaks were matched by Progenesis LC-MS, about 300 proteins were analyzed with significant difference.3. The different proteins was classified by Panther 9.0. In aspect of protein function, there were catalytic activity proteins, binding proteins, structural molecules, and so on in the biological process aspect, these proteins involved in metabolic process, cellular process, developmental process, and so on.4. About 80 different proteins with more than 1 matching peptides were analyzed by String 9.1. As the result showed, there were complex and close association among these proteins. The contact network can be broadly divided into three parts-endoplasmic reticulum stress reaction, transcriptional regulation of proteins and apoptosis.ConclusionLabel-free quantification method can provide references for the mechanism research in alcoholic liver and other clinical disease, could be well applied in medical study. |
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