Development of multiple enzymatic digestion, multidimensional HPLC coupled with mass spectrometry for proteomics analysis

• Main Authors: Jen-Ping Lin, 林振平
• Other Authors: Shui-Hui Chen
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/d7925d

碩士 === 國立成功大學 === 化學系碩博士班 === 92 === 　　This study is mainly based on developing a multiple enzymatic digestion method and a multi-dimensional chromatography system for the analysis of biological samples. It is hoped that using this technology, further protein analysis can be identified with an increasing variety and quantity of annotated proteins. Nowadays, proteomic research is one of the hot topics in the science research field. The great invention and improvement of mass spectrometric technology allow us to extend to the next level in the research. On the main focuses of this paper is on protein identification and analysis. 　　MALDI-TOF MS is one of the instrument that are used for protein analysis; its advantages include requiring small amounts of samples, short analyzing period, simple and convenient, as well as, high sensitivity. Before performing MALDI on the real sample, the proteins need to be digested into different sizes of molecules. Trypsin is the most common enzyme that is used in digesting proteins. However, due to the complexity of biological molecules, trypsin does have its limitations. If the enzyme has a hard time digesting the proteins, bad signals or even no signal would show on the screen. Consequently, different types of enzymes are added separately into each analyzing samples for digestion. Due to the fact that different enzymes have different characteristics, proteins would be digested at different digesting sites. The samples will then be detected with more signals than before. β-casein as well as rat placenta are both great examples in pointing out the advantages of using multiple enzymatic digestion method. 　　Besides MALDI-TOF MS, a multi-dimensional chromatography system would meet a problem when the quantity of analyzing protein increases with a constant number of fractionations. When this happens, the signals of proteins with tiny amount would be overlapped; therefore, we have come up with a multi-dimensional high performance liquid chromatography method to resolve the problem of overlapping signals. The original multi-D LC method starts its fractionation after the proteins are digested into peptides. The new method, however, performs fractionation at the protein level. The proteins are then digested with two enzymes followed by MALDI-TOF MS. Although an off-line stage is still presently being used for detection, shifting to an automatic system is being looked forward to. The data from MS are then being searched online via protein database by peptide mass mapping (PMM) technology. This multi-dimensional fractionation method is believed to be a useful way in finding more proteins when working with actual biological samples. The method is effective to increase for identifying those proteins in micro-units, even for some low-abundantly functional protein. Hopefully with all the conditions being tuned to optimal and automatic, this method could be used to analyze and identify cells and tissues.