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Development of Liquid Chromatography-mass Spectrometry Method and Its Application in Proteomic Analysis

Author: ZhouHu
Tutor: ZengZuo
School: Shanghai Institutes for Biological Sciences
Course: Biochemistry and Molecular Biology
Keywords: Liquid chromatography - mass spectrometry pH elution strong cation exchange liquid chromatography Membrane proteins Cholesterol gallstones 3T3-L1 fat cells Secreted proteins Lectin , concanavalin A affinity purification SD rats epididymis Glycosylation of proteomics
CLC: Q51-3
Type: PhD thesis
Year: 2007
Downloads: 1675
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Liquid chromatography-mass spectrometry is one of the most important analytical methods in proteome research. Although more proteins were identified by 2D LC-MS/MS method based on salt step elution than by 2-DE/MS method from human bile, it still suffered from complications mostly due to elution of the same peptides in the consecutive salt steps (peptide overlap), as well as from the interference of salt with the mass signal. Therefore, we have developed liquid chromatography-mass spectrometry methods based on pH gradient elution. Through these methods, peptides were separated efficiently by strong cation exchange chromatography. Utilizing these approaches, we analyzed samples from cell and tissue.To achieve sufficient separation and accurate identification, two-dimensional liquid chromatography mass spectrometry based on pH continuous online gradient (pCOG) or pH step online gradient (pSOG) was constructed. Results indicated that, the pH elution methods can obtain more unique peptides or proteins than salt elution ones, while in the same time increasing identification confidence and amino acid coverage. Also, more basic peptides were detected by pH elution method than by salt elution. By combining the pH elution and continuous gradient, the peptide cross-overlap was significantly minimized.Furthermore, the low salt concentration of the pH buffer used is compatible with mass spectrometry. Therefore, the pH continuous online gradient-strong cation exchange chromatography-mass spectrometry (pCOG-SCX-MS) method was established. The pCOG-SCX-MS can achieve better reproducibility and 10-fmol sensitivity. The pCOG-SCX-MS can also detect more trans-membrane peptides than conventional revere phase liquid chromatography-mass spectrometry (RP-MS). The pCOG-SCX-MS might serve as complementarities of and alternative to RP-MS in some instances.At the same time, liquid chromatography-mass spectrometry was used to analyze the samples from secretory proteins and SD rat epididymis. Label-free and cleavable isotope-coded affinity tag (cICAT) quantitative proteomic approaches were used to quantify the differential expression of secretory proteins from 3T3-L1 adipocytes with or without insulin stimulation. A total of 242 differentially expressed secretory proteins were quantified, including 55 down-regulated and 187 up-regulated secretory proteins by insulin stimulation. The concanavalin A binding method was used to purify the glycoproteins from caput and cauda of SD rat epididymis tissue. After cICAT quantification, differentially expressed glycoproteins were obtained, such as galectin and beta-hexosaminidase beta chain ets.

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CLC: > Biological Sciences > Biochemistry > Protein
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