| The number of proteins in complex biological systems and protein isomers generated by different modification can even reach to millions of kinds. Along with the progress of proteomics research, these complex proteomes with huge dynamic rangeand diverse physical and chemical properties have posed significant challenges for the exploration of life activities. High abundant proteins can be easily separated and identified, however, medium or low abundance proteins can’t be identified effectively. During the process of separation and identification, the strong signals from high abundant proteins may cover the signal from low abundant proteins, causing great disturbance to low abundance proteins. In addition, Proteome analysis also face many challenges, such as low sequence coverage of mass spectrometry(MS) data and poor quantitative stability. Therefore, researchers had proposed solutions from different angles in order to solve these problems.In proteomics, the shotgun method has been widely used in proteinsdetection. Proteins mixture can be digested to a mixture of peptides, and then mass spectra are adopted for sequence analysis. The experimental and theoretical spectra of a peptide were matched based on computer rules, and the position of identified peptides in protein sequence are located, thereby realizing the identification of proteins.The application of shotgun method has got rid of the limitations and shortcomings of the traditional gel separation technique, but it has also bro ught corresponding disadvantages. Through the adoption of shotgun, not only the high abundant peptides what can be easily detected by MSbut also the low abundance peptides what are difficult to detect were generated. The so-called low abundance does not mean that these peptides segment content is very low, they may simply be suppressed by huge number of high abundance peptides, or be left out during MS acquision due to the low ionization efficiency and limited MS sensitivity. However, even part peptide sequence or modified variants after translation can’t be effectively monitored for a variety of reasons, it does not imply that information carried by t he sequence is not important. O n the contrary, appropriate technology development is needed to find these missing peptides or proteins.The results of a large number of studies showed that low abundance peptides and proteins tended to carry important information or played irreplaceable roles in the biological world. Years of studies showed that in the aspect of disease treatment, low abundant peptides or proteins often served as markers for diagnosis, prognosis and treatment. At the same time, they may serve as targets for therapy. Therefore, the removal of high-abundant proteins and the enrichment of low abundant protein have become a research direction with great significance.The improvement of low abundant protein identification usually acted thro ugh three mechanisms:(1) consumption of high abundance protein;(2) enrichment of low abundance protein;(3) improvement in the sensitivity and accuracy of the analytical platforms. Wherein, improvement of separation and resolution of MS and chromatography technology usually isn’t cost-effective, and also is easily limited by instruments. Therefore, focus ing on the enrichment of low abundance proteins is the most common practice internationally, such as antibody immune removal, protein equalizer, modified proteins and nucleic acid binding proteins enrichment, etc. However, these methods have perference and poor versatility for complex samples. The current problem is the lack of simplification strategy for complex proteome system. This study is conducted from two aspects: first, taking advantage of the non-discriminatory reaction between amino reactive group or carboxyl reactive group and chemical group to achieve the purpose of high abundant proteins or peptides reduction in complex system; second, targeting to the enrichment of peptides containing a particular amino acid, thereby simplifying the system and reducing the matrix effect.The paper is divided into three parts, the first chapter sketched out the enrichment and identification technology for low-abundant proteins in proteome research, as well as the significance, status quo and progress of enrichment of peptides containing particular amino acid; meanwhile, the common methods used on low abundant protein identification, such as the removal of antibody immune, protein equalizer, modified proteins and nucleic acid binding, on this basis, this paper presented the content and objective of this study, i.e. high abundance protein removal method with strong versatility, and specific peptide enrichment strategy.In the second chapter, the exploration of the high-abundance proteins removal method was investigated. According to the amino reactive group or carboxyl reactive group, we optimized the concentration-speed relationship of combining amino or carboxyl group, t hus removing high-abundance proteins. In this experiment, peptides were treated as chemical molecules containing amino groups. When they were combined to react with amino reactive group or carboxyl reactive group, peptides from relatively high abundance proteins reacted with beads carrying special groups more quickly. In a conclusion, repeated coupling reaction can help to ultimately achieve the purpose of high abundance removal. My experiment is divided into two parts.Firstly, we studied the basis of this experiment, and examined three different methods using biological yeast cells; Secondly, we compared the extraction efficiency, the reproducibility of extraction methods, the yeast protein abundance obtained with different extraction methods, the linear relationship with opposite spectrogram number, and the comparison between protein abundance range that can be identified and theoretical value. With the experiment results in this part, we found that the extraction efficiency of NETN ultrasonication method was the highest, but it showed that SDS alkaline lysis and Urea ultrasonication had identified more proteins referring to mass spectrometry results. wherein, SDS alkaline lysis method had more obvious modification effect on peptides. Therefore, Urea ultrasonication is an ideal method for extraction.In the second part of study, Urea Ultrasonication was conducted to extract the proteins of yeast. In the experiment, cyanogen bromide beads were regarded as the main experimental subjects, and mass spectrometry analysis showed that a part of peptides were damaged after many times of binding, but the leveling effect between high abundance and low abundance was also quite significant.In the third chapter, along with the development of high-throughput proteomic research techniques, LC-MS/MS is widely used in the depth identification and quantitative analysis of target peptides and proteins. However, due to peak capacity constraint and matrix effect in liquid chromatography- mass spectrometry(LC-MS) analysis, only the effective protein sequence coverage was obtained, while a large number of implicit peptides and co-eluted peptides can seriously affect the accuracy of quantitative analysis. In order to simplify the analysis system and impro ve the accuracy of quantitative mass spectrometry analysis, this study explored selective enrichment methods on his-contained peptides, so as to establish a tandem analysis method for histidine peptides and quantitative proteome study. The results showed that, after 30 mins of reaction between Cu-bead and peptide, 1:1 proportion between peptide loading quality of sample and C u-beads(μg: ul) was an optimal condition, and his-peptides with good stability and high reproducibility can achieve the selective enrichment. In i TRAQ experiment with labeled proportion, his-peptides enrichment can significantly improve the accuracy of quantitative analysis. Therefore, his-peptides enrichment can be used as a simple yet effective strategy to simplify the complexity of the proteome, and improve the accuracy of quantitative proteomics research. |
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