Hydrogen atom scrambling in selectively labeled anionic peptides upon collisional activation by MALDI tandem time-of-flight mass spectrometry

Abstract

We have previously shown that peptide amide hydrogens undergo extensive intramolecular migration (i.e., complete hydrogen scrambling) upon collisional activation of protonated peptides (Jørgensen et al. J. Am. Chem. Soc. 2005, 127, 2785-2793. The occurrence of hydrogen scrambling enforces severe limitations on the application of gas-phase fragmentation as a convenient method to obtain information about the site-specific deuterium uptake for proteins and peptides in solution. To investigate whether deprotonated peptides exhibit a lower level of scrambling relative to their protonated counterparts, we have now measured the level of hydrogen scrambling in a deprotonated, selectively labeled peptide using MALDI tandem time-of-flight mass spectrometry. Our results conclusively show that hydrogen scrambling is prevalent in the deprotonated peptide upon collisional activation. The amide hydrogens . In comparison, deprotonated peptides have received much less attention, and the mechanistic details of their fragmentation behavior are less well understood. We and others have previously investigated the proton mobility in protonated peptides and proteins upon collision-induced dissociation (CID) [2][3][4][5][6][7][8][9][10][11][12]. Our studies were motivated by earlier reports claiming that CID could be used to determine site-specific incorporation of deuterium in the backbone amides of peptides that were deuterated in solution [13][14][15][16]. In these reports, it was suggested that the level of intramolecular migration of amide hydrogens ( 1 H/ 2 H) in the protonated, collisionally activated peptide was negligible. It is important to emphasize that negligible levels of hydrogen migration would dramatically improve the resolution of localizing deuterated sites by mass spectrometric analysis of peptides and proteins labeled in solution ( 1 H/ 2 H) exchange experiments. But recent results have demonstrated unequivocally that all backbone amide hydrogens are positionally randomized upon collisional activation in the gaseous protonated peptide (i.e., causing 100% hydrogen scrambling) [3,6,7,10]. This implies that protonation of amide nitrogens is a reversible process in the gaseous activated peptide, and that the mobile proton (or deuteron) samples all exchangeable sites before peptide bond cleavage. Interestingly, this also occurs in singly protonated peptides exhibiting a preferred site-selective cleavages at Asp residues, which are thought to be induced by a reduced proton mobility [6]. Deprotonated peptides are deficient in protons and it is thus reasonable to assume that such ions might exhibit a lower degree of hydrogen scrambling than their protonated counterparts. Furthermore, low-energy CID of deprotonated peptides often yield a fragment ion type (c-ion) that is observed only rarely in protonated peptides [17]. The c-ion is the N-terminal fragment ion that results from backbone N-C ␣ bond cleavage. As anionic peptides thus exhibit different fragmentation pathways (relative to their protonated counterparts) and they...

Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 2. Assessing Charge Site Location and Isotope Scrambling

“…The consideration of such differences between positively-charged and negatively-charged ions served as a motivation for the study presented here. Finally, it is noted that some have argued that collision-induced dissociation (CID) should not be used in HDX structural studies because of the potential for HD scrambling [10, 27] while others have suggested some utility for HDX-MS/MS studies [1, 28]. Here we extend the latter consideration by presenting an initial approach that determines the accessibility of hydrogens on model structures obtained from molecular dynamics simulations (MDS).…”

“…Therefore, effectively testing such assumptions requires access to accurate structures obtained from MDS. Finally, at the time, site-specific deuterium uptake information to aid model development could not be obtained due to problems associated with hydrogen-deuterium (HD) scrambling during the ion fragmentation process[9, 10]. …”

Measuring the Hydrogen/Deuterium Exchange of Proteins at High Spatial Resolution by Mass Spectrometry: Overcoming Gas-Phase Hydrogen/Deuterium Scrambling

“…[10][11][12]20 Interestingly, we have found that H/D scrambling is also prevalent in negative ion mode for CID of deprotonated peptides. 33 In this case, a "mobile deprotonation site" causes extensive internal rearrangement of deuterons initially located on the backbone amides. With the present knowledge of the prevalence of scrambling in protonated peptides and proteins, there is now a consensus that CID should not be used as an experimental tool to obtain information on the local deuterium uptake in solution.…”

Investigation of amide hydrogen back-exchange in Asp and His repeats measured by hydrogen (1H/2H) exchange mass spectrometry

“…Also, the selective labeling with an N-terminal half devoid of deuterium ensures a maximal sensitivity for detecting and measuring the level of gas-phase hydrogen scrambling. Using such peptides, we have demonstrated that CID [9,[18][19][20] cause extensive hydrogen scrambling, while electron capture dissociation (ECD) [11], electron transfer dissociation (ETD) [10] and MALDI in-sourcedecay [21] proceed with a negligible level of hydrogen scrambling. These fragmentation techniques are thus available as experimental tools in either a "bottom-up" [22] or a "top-down" [23][24][25] approach to determine site-specific deuterium levels of proteins from solution H/D exchange experiments provided that any excessive vibrational excitation is avoided prior to the fragmentation event.…”