Hydrogen/Deuterium Scrambling during Quadrupole Time-of-Flight MS/MS Analysis of a Zinc-Binding Protein Domain

Abstract

It remains an open question as to whether experiments involving collision-induced dissociation (CID) can provide a viable approach for monitoring spatially resolved deuteration levels in electrosprayed polypeptide ions. A number of laboratories reported the successful application of CID following solution-phase H/D exchange (HDX), whereas others found that H/D scrambling precluded site-specific measurements. The aim of the current work is to help clarify the general feasibility of HDX-CID methods, using a 22-residue zinc-bound protein domain (Zn-ZBD) as model system. Metal binding in Zn-ZBD should confer structural rigidity, and the presence of several basic residues should sequester mobile charge carriers in the gas phase. Both of these factors were expected to suppress the extent of scrambling. HDX was carried out by employing rapid on-line mixing, thereby mimicking conditions typically encountered in kinetic pulse-labeling studies. Quadrupole time-of-flight MS/MS of pulse-labeled Zn-ZBD provides high sequence coverage. However, the measured fragment deuteration levels do not correlate with the known H-bonding pattern of Zn-ZBD, suggesting the occurrence of extensive scrambling. Instead of showing a uniform distribution, the fragment ions reveal a distinct nonrandom pattern of deuteration levels. In the absence of prior information, these data could erroneously be ascribed to the presence of protected sites. However, the observed patterns clearly originate from other factors; possibly they are caused by modulations of the amide CID efficiency by kinetic isotope effects. It is concluded that scrambling does not represent the only conceptual problem in HDX-CID studies and that control experiments on uniformly labeled samples are essential for ruling out interpretation artifacts.

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

“…Bovine cyt c contains a total of 192 exchangeable hydrogens [53]. Hydrogen back exchange in the gas phase is negligible under the conditions used here [64].…”

“…The mass distribution of a broadened peak p after HDX may be approximated as a convolution of the protein's shifted mass profile f with a Gaussian distribution function D [64]. This can be expressed as…”

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

“…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].…”

Protein Structural Characterization by Hydrogen/Deuterium Exchange Mass Spectrometry with Top-down Electron Capture Dissociation

“…Our data showed that the deuteration level of Cterminal fraction (short z . ions) is higher than that of the Nterminal fraction (short c ions) after 24 h full H/D exchange, which is attributed that C-terminal fraction contains a highly flexible tail (residues 71-76) and N-terminal fraction contains two hydrogen-bonded β-strands (residues 1-7 and residues 10-17) and a tightly packed α-helix (residues [23][24][25][26][27][28][29][30][31][32][33][34]. The α-helix region showed the lowest deuteration level, and the turn (residues 8-11) on the surface of the protein that connects two adjacent β-strands showed very high deuteration level.…”

“…[20][21][22][23][24][25] However, others showed apparent scrambling resulted from CID in HDX/MS/MS studies. [26][27][28][29] Even though it has been reported that another threshold dissociation method, infrared multiphoton dissociation (IRMPD), 30,31 causes little or no deuterium scrambling in HDX-MS, 32 the labile modifications are lost prior to backbone cleavage, thus making it difficult to identify the modification site. Recently, Polfer and co-workers have reported abundant NH 3 and H 2 O losses from b fragments when employing IRMPD in HDX studies and they also reported that SORI CID (sustained offresonance irradiation collision-induced dissociation) 33 can cause more scrambling products and internal fragments.…”