| 1. Strategy optimization for identifying phosphopeptides by immobilized-metal ion affinity chromatography and mass spectrometryReversible phosphorylation is a key mode of signal transduction, a central mechanism in the regulation of protein expression and function, and able to regulate the majority of all cellular processes. Phosphoproteins are in relatively low abundance, negatively charged, and relatively hydrophilic. Therefore, both enrichment of phosphoproteins or phosphopeptides and sensitivity of the MS acquisition are prerequisites for efficient identification of phosphoproteins. As a fast, easy-to-use, and economic procedure, immobilized-metal ion affinity chromatography (IMAC) is recently used extensively for the research of phosphoproteomics. By using this method, researchers have been focusing their attentions on eliminating non-specific binding of non-phosphorylated peptides with acidic side-chains such as glutamic and aspartic acid residues. However, effect of nucleic acids in protein samples on phosphopeptide identification by IMAC- mass spectrometry (MS) strategy has not described in phosphoproteomic studies. So far, two IMAC strategies adopted by researchers are ME-IMAC (with methyl esterification of peptide samples before IMAC enrichment) and Non-ME-IMAC (without methyl esterification of peptide samples before IMAC enrichment). In this study, by comparing ME-IMAC strategy and non-ME-IMAC strategy, we firstly pointed out that ME-IMAC strategy could cause adsorptive losses easily and complicate the MS analysis and data interpretation, but Non-ME-IMAC strategy was a simpler and more efficient strategy for phosphopeptide enrichment. Then the experimental conditions of non-ME-IMAC strategy were optimized in detail. We found that the best loading/washing buffer for Non-ME-IMAC was 8%ACN/0.3%TFA, and the best eluting buffer for Non-ME-IMAC was 0.2mol/L phosphate solution (pH 8.0). Besides we firstly point out that SSNAs have significantly adverse effects to phosphopeptides enrichment by IMAC, have strong affinities to the matrix of IMAC, and have significantly adverse effects (inhibition ratio >70%) on phosphopeptides enrichment by IMAC. In this study, we found a simple and efficient method-"ACN precipitation" to remove nucleic acids to reduce the adverse effects and the enrichment ratio could increase more than about 200%. This study will be a good reference to more and more researchers in phosphoproteomics.2. The aging-related proteome analysis of splenic lymphocytes in senescence-accelerated mouseAging is associated with a wide of spectrum of degenerative diseases, including Alzhimer's disease, Parkinson's disease and disorder of immunological functions. Senescence-accelerated mice belong to inbred strain mouse, and many age-related functional disorders appear with their increasing age. Besides, many pathobiological phenotypes of senescence-accelerated mice are similar with aging human. In order to detect differential proteome that may involved in aging or aging-related diseases between SAMP8 and SAMR1, the splenic proteomes from 9-month-old SAMP8 and SAMR1 were prepared and separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). As a result, twenty one differentially expressed protein spots were detected. The aged SAMP8 mice showed a large increase of over 2-fold in the expression of thirteen spots, and the expression of eight spots was elevated over 2-fold in SAMR1 compared to SAMP8. The differential spots were submitted to peptide mass fingerprint (PMF) analysis by MALDI-TOF-MS, and then using the software MASCOT to search the protein database online. A protein named "Annexin I", which was up-regulated in the splenic lymphocytes of SAMP8, was validated by Western blot. The functional implications of other differentially expressed proteins involved in aging or aging-related diseases were discussed. |
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