Improvement of peptide detection by LC-ESI-MS；Focus on solvent compensation

• Main Authors: Shih-Ying Tai, 戴詩穎
• Other Authors: Kuo-Lung Ku
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/96077867680488158156

碩士 === 國立嘉義大學 === 應用化學系研究所 === 101 === Peptides of digested protein are not fully observed by MS-based proteomic methods, such as LC-ESI-MS. A peptide was classified as proteotypic when the number of observation is 100% of all injection numbers. Increasing the number of peoteotypic peptides will therefore give more accurate information for protein identification. For the detected number of peptides, electrospray ionization efficiency has a great influence. And the factor divide into the analyte nature and instrument parameters. In the present study, we aim at discuss analyte property in fixed LC-ESI-MS parameters conditions. However, for the separation of peptides of different polarity, different analytic solvent environment could cause different ionization efficiency. Therefore, the amount of the proteotypic peptides detected in ESI-MS is usually restricted by the low ionization efficiency of those highly water soluble peptides presented in the digested mixture. Here, we provided a method, post-column solvent compensated device in LC-ESI-MS for increasing the detection number of proteotypic peptides via adjust the gradient LC eluents to a specific relative nonpolar solvent composition. In order to maximize the ionization efficiency for peptides with various polarities, the ratio of make-up solvent based on authentic peptides is 50% MeOH. The method is demonstrated by uthentic peptides (CALNNPKEK, LSA and GLGLG) and enzymatic digested model proteins (BSA, ovalbumin, and β-lactoglobulin). The MS peak area of CALNNPKEK detected under make-up condition (make-up to 50% MeOH) was 2.04 times to that of the non-make-up condition. And the followed species LSA and GLGLG were 1.65 and 1.26 times to the non-make-up ones, respectively. Those data revealed that the ionization efficiencies of authentic peptides in LC-ESI-MS were all improved. The detected number of proteotypic peptides of the enzymatic digested model proteins increased by the polarity of the original proteins.