Imidazolium Compounds as Internal Exchange Reporters for Hydrogen/Deuterium Exchange by Mass Spectrometry

Abstract

Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a powerful tool for protein structure analysis that is well suited for biotherapeutic development and characterization. Because HDX is strongly dependent on solution conditions, even small variations in temperature or pH can have a pronounced effect on the observed kinetics that can manifest in significant run-to-run variability and compromise reproducibility. Recent attention has been given to the development of internal exchange reporters (IERs), which directly monitor changes to exchange reaction conditions. However, the currently available small peptide IERs are only capable of sampling a very narrow temporal window and are understood to exhibit complex solution dependent exchange behavior. Here we demonstrate the use of imidazolium carbon acids as superior IERs for HDX-MS. These compounds exhibit predictable exchange behavior under a wide variety of reaction conditions, are highly stable, and can be readily modified to exchange over a broad temporal window. The use of these compounds as IERs for solution based HDX-MS could considerably extend the utility of the technique by allowing for more robust empirical exchange correction, thereby improving reproducibility.

Developments in Hydrogen/Deuterium Exchange Mass Spectrometry

“…Lastly, we want to briefly bring attention to some articles that are important to HDX MS in their own right, which may guide and drive some of the future development of the field outside of the broad areas of LC, MS, or data processing. These articles include use of HDX MS in biopharma to monitor changes due to chemical modification (e.g., methionine oxidation), decoupled automation wherein sample preparation is a separate automation process from automated LC–MS, deamidation and protein stability, optimizing various points along the experimental workflow, including and exploiting PTMs during analysis, internal standards that report on the exchange conditions and back-exchange along the analysis pathway in order to correct for experimental condition fluctuations, capillary electrophoresis for separating coexisting protein states that are all deuterated together, and corrections to account for buffer additives that can alter the intrinsic rate of exchange …”

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“…Robust comparative studies require careful matching of solution exchange conditions to minimize interlab variability. 102 , 131 , 409 , 632 …”

“…Murphree et al described a series of benzimidazolium compounds that exchange solely at a single acidic carbon on a wide time scale that is relevant for HDX-MS studies. 102 The exchange of the imidazolium group is purely base-catalyzed, so the exchange rate is linearly correlated with solution pH. While further developments are necessary to establish more reliable and versatile IERs, such standards may be valuable for addressing interday and even interlab variability in HDX-MS studies by providing users with a more robust method of detecting and correcting for variation in solution conditions during the deuterium labeling step.…”

Rapid Assessment of Pepsin Column Activity for Reliable HDX-MS Studies

“…MS/MS data were obtained using a Thermo LTQ-Orbitrap as described previously. 14 Peptide assignments were made using data-dependent acquisition MS/MS analyzed with Byonic (Protein Metrics) with a score cutoff of 100. Digestion efficiency tests were collected on a second custom HDX-MS system coupled to a Waters Synapt G2-Si as described previously.…”

“…MS/MS data were obtained using a Thermo LTQ-Orbitrap as described previously . Peptide assignments were made using data-dependent acquisition MS/MS analyzed with Byonic (Protein Metrics) with a score cutoff of 100.…”