Hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS) has become a powerful tool to study protein and protein complex conformations, at biological concentrations (usually at pico-molarity level).
High-resolution mass analysis (such as Fourier transform ion cyclotron resonance mass spectrometer) is particularly advantageous for the HDX MS method, by accurately assigning the peptide fragments after protease digestion and in resolving overlap of peptide isotopic distributions, both before and after HDX MS. Chapter 1 introduces the theory of the instrument for mass measurement, ionization methods for biological samples. An introduction of several biophysical methods to study protein conformation and protein-ligand interactions is also covered. The last portion of this chapter gives an overview of the HDX MS method, its history, development and applications.
In Chapter 2, a new enzyme, protease type XIII from Aspergillus saitoi, is characterized. This enzyme prefers to cleave on the C-terminal end of basic amino acids (i.e., histidine, arginine and lysine). Under positive-ion electrospray conditions, basic amino acid side chains are protonated, to yield abundant positive ions during ESI, resulting in increased signal-to-noise ratio for protease type XIII cleaved fragments.
Back exchange is the single biggest challenge for HDX MS and results in loss of information, because with back exchange, a fast exchanged D quickly exchanges back to H after quench and appears to be no exchange. We have been addressing the back exchange problem by reducing H2O content during peptides separation with salts. We replaced high performance liquid chromatography (HPLC) (H2O mobile phase) by on-line supercritical fluid (a state between gas and liquid) chromatography (SFC).
In parallel with SFC, Chapter 3 describes a fast liquid chromatography method to reduce back exchange. Back exchange is reduced by at least 25% compared to conventional LC separations in the HDX community. A sham digestion method is employed to prove that no backbone hydrogen back exchange occurs during digestion and it mainly occurs during following LC separations.
Chapter 4 describes a Python program to sort data for each fragment under different conditions, and plot time-course deuterium uptake profiles and bar graphs for comparison. This programming demonstrates dramatically improved analysis accuracy and efficiency (analysis time reduced from several days to less than 10 min!).
Certain cancers such as gastrointestinal stromal tumors (GISTs) exhibit elevated expression and/or activating mutations of the receptor tyrosine kinase, KIT. Like many kinases, KIT has a flexible kinase insertion domain (KID).
Chapter 5 discusses the use of solution-phase HDX experiments to verify that the KID has no conformational effect on KIT either in the presence or absence of sunitinib. By comparing the conformation of a sunitinib resistance mutant (D816H) with wild-type KIT, the drug resistance mechanism for KIT is elucidated.
The RAGE (Receptor for Advanced Glycosylation End Products) protein has been proposed to be involved in the active transport of beta-amyloid protein across the blood-brain barrier, which is thought to be one of the major contributing factors of Alzheimer’s disease. Consequently, RAGE is being targeted to prevent the passage of amyloid beta.
Chapter 6 discusses use of solution-phase HDX to study the conformational changes in RAGE induced by the presence of glycosylation. A region of binding beta-amyloid was found to be more flexible in the BacMam expressed RAGE than the E. coli expressed RAGE due to the opening effect by glycosylation on BacMam-RAGE. This change in structure has great potential to facilitate the design of therapeutics.
As an effector, L-leucine binds on the regulatory domain and causes allosteric change on the active site of MtIPMS. Chapter 7 talks about use of HDX MS to map the allosteric regulation pathway in the IPMS protein upon L-leucine binding. An L-leucine-insensitive mutant Y410F MtIPMS was used to verify the inhibition transduction pathway. These results demonstrate one of the first reports of an experimentally mapped allosteric mechanism in a protein of this size. These results will facilitate the design of new anti-TB therapeutics. A manuscript of these results has been recently submitted to Biochemistry. (Abstract shortened by UMI.)