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Mass Spectrometry Based Method Development for Elucidation of Protein Sequences, Structures and Post Translational Modifications

  • Author(s): Sun, Qingyu
  • Advisor(s): Julian, Ryan R
  • et al.
Abstract

The advanced development of mass spectrometry (MS) makes MS a powerful technique for proteomics study. The increasing demands for proteomics study stimulate creation of more applicable MS-based methods. This dissertation focuses on development of novel MS-based methods to characterize three different aspects of proteins: primary sequence, post-translational modifications (PTMs) and three-dimensional (3D) structure.

The first part (Chapter 2) is focused on primary sequence characterization. A novel technique radical directed dissociation (RDD) is developed for peptide gas phase fragmentation. RDD is recognized as a "charge-remote" dissociation in which RDD backbone fragments are mediated by &betaC-H bond dissociation energies (BDEs) of all 20 amino acids. Therefore, RDD fragmentation of peptides with known sequences is predictable. This discovery indicates RDD is promising for proteomics study in terms of significantly improving the confidence level of peptide identification.

The second part covers MS-based method development for PTM characterization. Chapter 3 describes using RDD to rapidly map iodinated tyrosines in intact proteins. The iodinated tyrosines are identified in the portions of a protein where RDD fragments are frequently located. The only limitation is that when multiple tyrosines are located in the identified iodinated region, it could be challenging to differentiate the iodination states between adjacent tyrosines. In Chapter 4, iodination chemistry combined with tandem MS is used to localize acid-labile histidine phosphorylation sites in peptides. Phosphorylated histidines are immune of iodine-labeling whereas unmodified histidines are not. After iodination, labile histidine phosphates can be removed by acid treatment to yield free histidines, which can be easily identified by MS/MS. This new method provides a pathway forward for analyzing histidine phosphorylation in complex systems.

The last part is aimed at protein conformational study using selective noncovalent adduct protein probing-mass spectrometry (SNAPP-MS). SNAPP-MS employs 18-crown-6 ether (18C6) as a probe of lysines and the numbers of 18C6s attached to proteins reveal protein solution phase structural information. In Chapter 5, SNAPP-MS is used to study the dynamic structural interchanging in Lymphotactin (Ltn). SNAPP successfully encodes the interchanging transition state of Ltn, which provides valuable information for interpreting Ltn biological functions.

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