Protein Conformation in Amorphous Solids by FTIR and by Hydrogen/Deuterium Exchange with Mass Spectrometry

Abstract

Solid-state hydrogen/deuterium exchange (ssHDX) with electrospray ionization mass spectrometry (ESI-MS) and Fourier transform infrared (FTIR) spectroscopy were used to assess protein conformation in amorphous solids. Myoglobin, lysozyme, beta-lactoglobulin, ribonuclease A, E-cadherin 5, and concanavalin A were co-lyophilized with carbohydrates (trehalose, raffinose, and dextran 5000), linear polymers (polyvinyl alcohol and polyvinyl pyrrolidone) or guanidine hydrochloride (negative control). For ssHDX, samples were exposed to D2O vapor at 33% relative humidity and room temperature, and then reconstituted at low temperature (4 degrees C) and pH 2.5 and analyzed by ESI-MS. Peptic digestion of selected proteins was used to provide region-specific information on exchange. FTIR spectra were acquired using attenuated total reflectance. FTIR and ssHDX of intact proteins showed preservation of structure by raffinose and trehalose, as indicated by FTIR band intensity and protection from exchange. ssHDX of peptic digests further indicated that these protective effects were not exerted uniformly along the protein sequence but were observed primarily in alpha-helical regions, a level of structural resolution not afforded by FTIR. The results thus demonstrate the utility of HDX with ESI-MS for analyzing protein conformation in amorphous solid samples.

Investigating the influence of effective parameters on molecular characteristics of bovine serum albumin nanoparticles

“…Extended or random coils 1630-1635 ---Although FT-IR can detect gross changes in protein secondary structure, the method lacks sufficient resolution to detect more subtle structural changes [30]. So here further structural details were confirmed by FT-Raman spectroscopy to acquire information on protein structure.…”

Predicting Protein Aggregation during Storage in Lyophilized Solids Using Solid State Amide Hydrogen/Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS)

“…16−19 In ssHDX-MS, lyophilized samples are exposed to D 2 O in the vapor phase at controlled relative humidity (RH) and temperature. Samples are reconstituted under quenched conditions and subjected to MS analysis with or without proteolytic digestion.…”

Localized Hydration in Lyophilized Myoglobin by Hydrogen–Deuterium Exchange Mass Spectrometry. 1. Exchange Mapping

“…In recent studies, our group has adapted hydrogen-deuterium exchange with mass spectrometric analysis (HDX-MS) to proteins in the solid state 21–24 , providing higher resolution information on protein structure and matrix interactions. HDX-MS has been widely used to assess solvent exposure and dynamics for proteins in solution 25–27 .…”