Study Of New Nanoparticles-based Pre-treatment Methods And Their Applications In Proteomics Research

Nanoparticles have unique physical and chemical properties due to their size and shape. Some promising protocols based on nano-particles materials have been used to improve the analyses of biological samples. In our study, two new nanoparticles-based pre-treatment methods, such as the strategy based on magnetic nanoparticles coated with zirconium phosphonate for the enrichment of phosphopeptides and the method for desalting directly on MALDI-TOF MS target based on the absorption of peptides with Fe3O4/TiO2 nanoparticles, have been developed. And in another work, the effect among gradient elution time, repeated runs and narrow mass range scanning on achieving the completeness of proteome has also been investigated.Protein phosphorylation, as one of the most important post-translational modifications in mammalian, regulates many important biological processes, including cell proliferation, differentiation, metabolism, communication, and signal transduction. Global analysis of protein phosphorylation in cell or tissues is very significant for exploring these critical processes. However, the analysis of phosphopeptides is still a challenge such as low ionization efficiencies of phosphorylated peptides, and signal suppression by nonphosphopeptides in MS. Therefore, enrichment of phosphopeptides is a very key step for analysis of phosphoproteins. The detection of phosphopeptides has been improved by many enrichment approaches that reduce sample complexity and increase the relative concentration of phosphopeptides, but there are still requirements for developing novel methods, techniques, and affinity materials for large scale phosphoproteome analysis. In this study, novel phosphopeptide enrichment materials and approaches have been developed for phosphoprotein analysis.A novel strategy based on magnetic nanoparticles coated with zirconium phosphonate for the enrichment of phosphopeptides has been developed for the first time in this study. The highly effective enrichment of phosphopeptides by the nanoparticles are attributed to the strong interaction between phosphopeptides and zirconium phosphonate attached to the nanoparticles, and enhanced by the high trapping capacity of nanoparticles due to their extremely small sizes. With a standard phosphopeptide (FLpTEYVATR, m/z = 1179.55), the loading capacity of the nanopaticles is measured to be 141.2 pmol/mg. Under optimized experimental conditions, 1×10-9 M phosphopeptides in 50μL from tryptic digest ofβ-casein could be enriched and identified successfully. Reliable results were also obtained for 1×10-8 M phosphopeptides in 50μL from tryptic digest ofβ-casein in the presence of nonphosphopeptides from tryptic digest of BSA over 20 times in concentration. The performance of nanoparticles for use in a real sample was further demonstrated by enriching the SCX fraction of tryptic digest of protein extract from Chang liver cells as a model sample with the nanoparticles. Experimental results show that the nanoparticles can be easily and effectively used for enrichment of phosphopeptides in low concentration. Most importantly, our approach is more compatible with commonly used SCX strategy than Fe3+-IMAC. The proposed method thus has great potential for future application into large scale phosphoproteome research.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), combined with 2-DE, is becoming a classic technique for the proteome analysis. Some problems exist in the analysis of complex samples by MALDI-TOF-MS. For example, urea or ammonium bicarbonate commonly used to denature the proteins or to regulate th pH of used buffers, will affect the form of co-crystals among analytes and matrix, and thereby seriously degrade the quality of mass spectra. To meet the requirements of high-throughout proteome analysis, lots of salt-tolerant matrices have been investigated, and a number of methods for the sample pretreatment have been also developed. Despite the effective removal of contaminants by above-mentioned methods, the poor tolerance of contaminants with high concentration, sample loss, and laborious handling process have still been encountered. So it is necessary to establish an effective method for sample desalting. In this study, a novel method of directly desalting on MALDI-TOF MS target has been established for the application in a large scale proteomics studies based on the absorption of peptides with Fe3O4/TiO2 nanoparticles. After optimization of experimental conditions, such as the amounts of used nanoparticles and washing solutions, 100 fmol myoglobin digested by trypsin in 10 M urea solution could be identified successfully, and the reliable result could also be obtained for 10 fmol myoglobin in 3 M urea solution. The experimental results show that the method developed can be easily and effectively used for the protein identification in high salt buffere and has great potential for the large scale proteomics studies in the future. In another work, to study the effect of gradient elution time, repeated runs and narrow mass range scanning on the identification rates of proteome in a sample by a shotgun method, yeast lysate was used as a model sample and a series of experiments were performed on a nano-scale capillary reversed-phase chromatography interfaced to LCQ mass spectrometer. Experimental results show, to some extent, that totally identified unique peptides, which finally result in the identification of protein groups, increase remarkably with longer gradient time, repeated runs of analyzing a sample and segmented mass ranges, but employing segmented mass ranges is more effective than using longer gradient time or simply repeated runs for improving coverage of yeast proteome. The conclusion also suggests that employing complementary analytical strategies be a better choice in a large-scale proteomic analysis.