Gas-Phase Hydrogen/Deuterium Scrambling in Negative-Ion Mode Tandem Mass Spectrometry

Abstract

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS) has become a powerful method to characterize protein conformational dynamics. Workflows typically utilize pepsin digestion prior to MS analysis to yield peptide level structural resolution. Tandem mass spectrometry (MS/MS) can potentially facilitate determination of site-specific deuteration to single-residue resolution. However; to be effective, MS/MS activation must minimize the occurrence of gas-phase intramolecular randomization of solution-generated deuterium labels. While significant work has focused on understanding this process in positive-ion mode, little is known about hydrogen/deuterium (H/D) scrambling processes in negative-ion mode. Here, we utilize selectively deuterated model peptides to investigate the extent of intramolecular H/D scrambling upon several negative ion mode MS/MS techniques, including negative-ion collision induced dissociation (nCID), electron detachment dissociation (EDD), negative-ion free radical initiated peptide sequencing (nFRIPS), and negative ion electron capture dissociation (niECD). H/D scrambling was extensive in deprotonated peptides upon nCID and nFRIPS. In fact, the energetics required to induce dissociation in nCID are sufficient to allow histidine C-2 and C β hydrogen atoms to participate in the scrambling process. EDD and niECD demonstrated moderate H/D scrambling with niECD being superior in terms of minimizing hydrogen migration, achieving ~30% scrambling levels for small c-type fragment ions. We believe the observed scrambling is likely due to activation during ionization and ion transport rather than during the niECD event itself.

Fenton-Chemistry-Based Oxidative Modification of Proteins Reflects Their Conformation

“…For this reason, the best results are often obtained using automated sample preparation and handling, which allows better precision and accuracy than human-level control over the experiment. Finally, due to the mobilisation of protons (or deuterons) in gas-phase peptides under high-energy conditions, the most common fragmentation technique in mass spectrometry—collision-induced dissociation—causes randomisation (‘scrambling’) of labelling sites, which limits the resolution of the method to the peptide level [ 21 , 22 ].…”

Avoiding H/D Scrambling with Minimal Ion Transmission Loss for HDX-MS/MS-ETD Analysis on a High-Resolution Q-TOF Mass Spectrometer

“…However, a complicating factor for the implementation of HDX-MS/MS methodologies is the risk of intramolecular hydrogen/deuterium migration caused by excessive collisional heating of the analytes in the gas phase. Such heating results in a positional randomization of the isotopic labeling, a process termed hydrogen scrambling [35][36][37][38][39][40][41][42] . Thus, negligible hydrogen scrambling is a prerequisite for using HDX-MS/MS as tool for improving the resolution.…”

“…Thus, negligible hydrogen scrambling is a prerequisite for using HDX-MS/MS as tool for improving the resolution. In this regard, it should be noted that collision-induced dissociation (CID) cannot be used as a fragmentation method of protonated peptides to improve the resolution by HDX-MS/MS as it causes extensive scrambling in both positive and negative mode MS 35,[37][38][39][40][41][42][43][44][45] . However, prompt gas-phase fragmentation techniques such as MALDI-ISD, electron-based fragmentation (ECD/ETD), and more recently ultraviolet photo dissociation (UVPD) occur without inducing hydrogen scrambling [46][47][48][49][50] .…”

“…However, a complicating factor for the implementation of HDX-MS/MS methodologies is the risk of intramolecular hydrogen/deuterium migration caused by excessive collisional heating of the analytes in the gas phase. Such heating results in a positional randomization of the isotopic labeling, a process termed hydrogen scrambling. − Thus, negligible hydrogen scrambling is a prerequisite for using HDX-MS/MS as a tool for improving the resolution. In this regard, it should be noted that collision-induced dissociation (CID) cannot be used as a fragmentation method of protonated peptides to improve the resolution by HDX-MS/MS as it causes extensive scrambling in both positive- and negative-mode MS. ,− However, prompt gas-phase fragmentation techniques such as MALDI-ISD, electron-based fragmentation (ECD/ETD), and more recently ultraviolet photodissociation (UVPD) occur without inducing hydrogen scrambling. − Importantly, hydrogen scrambling is likely to occur in the ion source and in high-pressure ion transmission stages prior to fragmentation unless the level of vibrational excitation is carefully kept in check.…”

“…Such heating results in a positional randomization of the isotopic labeling, a process termed hydrogen scrambling. − Thus, negligible hydrogen scrambling is a prerequisite for using HDX-MS/MS as a tool for improving the resolution. In this regard, it should be noted that collision-induced dissociation (CID) cannot be used as a fragmentation method of protonated peptides to improve the resolution by HDX-MS/MS as it causes extensive scrambling in both positive- and negative-mode MS. ,− However, prompt gas-phase fragmentation techniques such as MALDI-ISD, electron-based fragmentation (ECD/ETD), and more recently ultraviolet photodissociation (UVPD) occur without inducing hydrogen scrambling. − Importantly, hydrogen scrambling is likely to occur in the ion source and in high-pressure ion transmission stages prior to fragmentation unless the level of vibrational excitation is carefully kept in check. Indeed, standard instrumental settings in normal proteomics experiments are often relatively harsh with respect to vibrational excitation to ensure maximal sensitivity (i.e., optimal ion desolvation, ion transmission, and fragmentation).…”