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MALDI in-source decay (ISD) allows for the rapid top-down terminal sequencing of intact proteins.The ESI LC-MS/MS bottom-upmethodis the traditional approach topcptide mapping and MS sequencing methodologies.Both of them arepowerful tools to characterize sequence modifications.These modifications could be either cell-based post-translational modifications,or intentional chemical modifications employed for drug development.The objective of this study is to utilize MALDI-ISD top-down and ESI LC-MS/MS bottom-up method to characterize unknown drug binding siteson therapeutic protein target.The initial work of this study is to utilize theability of intact mass detection and ISD method by MALDI TOF/TOF to simply and efficientlymonitor the mass shift on the intact and N-terminus of the protein.Through the specific 126 Damass shift on inz+2 ions of the protein iodination of histidine and tyrosine amino acidswas detected.To further confirmthe modifications,the protein was digested by trypsin and analyzed by ESI LC-MS/MS.