The Method Development For Microscale Sample Preparation And Mass Spectrometry Analysis In Proteomics Research

Proteomics depending on mass spectrometry is a power weapon for discovering clinical pathological information. In this paper, we aimed at the preparation of peptides from microscale sample for mass spectrometry analysis in the field of basic and clinical proteomics research. There are two parts in this paper:1) Methodological study for protein extraction from formalin-fixed and paraffin-embedded tissue and protein identification by mass spectrometry; 2) cell separation and extraction of mouse live.Clinical pathological samples can be stored for a long time by FFPE. These clinical samples containing complete clinical information and are prefect specimen for pathological proteomics research and disease marker discovery. High-throughput proteomics analysis of FFPE tissues is a power approach for finding the biologically and clinically valuable proteins related to disease diagnosis.In the first part of this paper, we construct the extracting method for FFPE tissues. Microscale protein extracted from the FFPE was digested by in gel digestion or in-situ digestion, and the peptides were identified by mass spectrometry. The result displayed no difference between the FFPE mouse liver and fresh mouse liver. This result indicated that the FFPE process had no effect for expression profile identification, and normal proteomic analysis process was suitable for FFPE mouse liver.Polyacrylamide gel is a prefect medium for avoiding pollution of small molecules and loss of sample in the digesting process for microscale protein. Two ways for immobilizing proteins into the gel were compared, one way was electrophoresis separation in SDS-PAGE for a short time, and the other was to immobilize proteins into the gel block in-situ directly. In a mass spectrometry analysis for 90 minutes,2931 proteins were identified from the peptides of 10 μg total Hela cell lysis by in gel digestion. With the same experiment condition,2301 proteins were identified from fresh mouse liver and 2264 proteins from FFPE mouse liver. This result indicated that our extracting method for FFPE was feasible and in gel digestion was a suitable way for digesting of microscale protein.The in-situ gel digestion was also investigated to identify the same samples above.The feature of this method was that during the polyacrylamide gel solidification process, the samples was immobilized at the same time. Washing and trypsin digesting steps were the same as in gel digestion. Thus, compared with in gel digestion, in-situ gel digestion process was simple, and suitable for in parallel process. Based on this method, 2327 proteins were identified from total Hela cell lysis,2164 from fresh mouse liver and 2077 from FFPE mouse liver. The identified amount was a little less than in gel digestion, but the amount and category of proteins were more stable, indicated that this method was much more suitable for label-free quantitative proteomics.According to our result, the FFPE process had no negative effect for protein digestion. According to the total ion chromatogram, we found that the peptides eluting sequences were the same, indicated that the FFPE process had no effect to the total hydrophobicity of peptide mixture. These results indicated that the FFPE tissues can contain the entire proteomics information of clinical samples, thus they are valuable for high-throughput proteomics analysis and potential biomarker discovery.In the second part of this paper, we aimed at the cell separation of mouse liver for the following proteomics research of cell secretory proteins and liver subtype cells. We improved the Seglen method for digesting the mouse liver into mixed suspension cells. Cell suspension was prepared by cell scribble, then the HC and NPC cells were separated by differential centrifugation. Morphologic observation and immunoblotting results indicated that the HC and NPC cells were separated successfully.