Probing qualitative conformation differences of multiply protonated gas-phase proteins via hydrogen/deuterium isotopic exchange with water-d2

115

Dissociation of gas-phase protonated protein dimers into their constituent monomers can result in either symmetric or asymmetric charge partitioning. Dissociation of ␣-lactalbumin homodimers with 15ϩ charges results in a symmetric, but broad, distribution of protein monomers with charge states centered around 8ϩ/7ϩ. In contrast, dissociation of the 15ϩ heterodimer consisting of one molecule in the oxidized form and one in the reduced form results in highly asymmetric charge partitioning in which the reduced species carries away predominantly 11ϩ charges, and the oxidized molecule carries away 4ϩ charges. This result cannot be adequately explained by differential charging occurring either in solution or in the electrospray process, but appears to be best explained by the reduced species unfolding upon activation in the gas phase with subsequent separation and proton transfer to the unfolding species in the dissociation complex to minimize Coulomb repulsion. For dimers of cytochrome c formed directly from solution, the 17ϩ charge state undergoes symmetric charge partitioning whereas dissociation of the 13ϩ is asymmetric. Reduction of the charge state of dimers with 17ϩ charges to 13ϩ via gas-phase proton transfer and subsequent dissociation of the mass selected 13ϩ ions results in a symmetric charge partitioning. This result clearly shows that the structure of the dimer ions with 13ϩ charges depends on the method of ion formation and that the structural difference is responsible for the symmetric versus asymmetric charge partitioning observed. This indicates that the asymmetry observed when these ions are formed directly from solution must come about due either to differences in the monomer conformations in the dimer that exist in solution or that occur during the electrospray ionization process. These results provide additional evidence for the origin of charge asymmetry that occurs in the dissociation of multiply charged protein complexes and indicate that some solution-phase information can be obtained from these gas-phase dissociation experiments. (J Am Soc Mass

Section: Gas-phase H/d Exchange Of Protein Complexesmentioning

confidence: 99%

Further studies on the origins of asymmetric charge partitioning in protein homodimers

Jurchen

García

Williams

2004

115

“…Although H/D exchange reagents can access all labile hydrogen atoms of small peptides, the accessibility of exchange reagents toward labile hydrogen sites of larger peptides and proteins is conformation dependent [1], thus the rate of H/D exchange reactions and the number of hydrogen atoms exchanged can be used as a structural probe [2]. H/D exchange and mass spectrometry can be combined to study both gas-phase and solutionphase H/D exchange reactions [3][4][5][6][7][8][9], and the kinetics of H/D exchange can be utilized to infer the presence of distinct conformations of both solution-phase [10 -12] and gas-phase peptide and protein ions [13]. For example, McLuckey et al studied the gas-phase H/D exchange of bradykinin [M ϩ H] ϩ ions (amino acid sequence RPPGFSPFR) with DI and D 2 O, and they interpreted the observed bimodal distributions of product ions as evidence for two non-interconverting ion conformations having different reactivities toward deuterating reagents [14].…”

mentioning

confidence: 99%

A mechanistic study of the H/D exchange reactions of protonated arginine and arginine-containing Di- and tripeptides

Huang

Marini

McLean

et al. 2009

The gas-phase H/D exchange reactions of arginine (R) and arginine-containing di-and tri-peptide (gly-arg (GR), arg-gly (RG), gly-gly-arg (GGR), gly-arg-gly (GRG) and arg-gly-gly (RGG)) [M ϩ H] ϩ ions with deuterated ammonia (ND 3 ) were investigated by using Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR), ion mobility-mass spectrometry (IM-MS), ab initio and density functional theory-based molecular orbital calculations and molecular modeling. Three exchanges are observed for arginine and argininecontaining tri-peptide [M ϩ H] [15]. Valentine and Clemmer used ion mobility-mass spectrometry to examine the conformation dependence of H/D exchange reactions, and suggested that ion conformation has a significant effect on H/D exchange [16,17].Compared with the intensive investigations carried out on the conformational characterization of peptides and proteins using solution-phase H/D exchange reaction chemistry, the corresponding gas-phase studies are much less comprehensive. The key question regarding gas-phase H/D exchange is: does the "solvent molecule," i.e., H/D exchange reagent, sample the entire "available surface area" of the peptide/protein ion, or is H/D exchange limited by other factors? For example, are all solution phase labile hydrogen atoms exchangeable in the gas phase and are the reactivities of all exchangeable hydrogen atoms similar? Dookeran and Harrison studied the H/D exchange reactions of 18 naturally occurring amino acids and selected di-and tripeptides with ND 3 , and reported that amino acid [M ϩ H] ϩ ions containing hydroxyl, carboxyl, and amine side chains exchange all labile hydrogen atoms, whereas the side-chain hydrogen atoms of glutamine, asparagine, and histidine [M ϩ H] ϩ ions exchange less readily, and tyrosine and arginine [M ϩ H] ϩ ions do not undergo significant exchange [18]. In a separate study, Lebrilla et al. found that the carboxylic acid group of glycine and aliphatic amino acid [M ϩ H] ϩ ions (alanine, valine, leucine, isoleucine, and proline) undergo H/D exchange three to 10 times faster than the NAddress reprint requests to Dr.

“…Recently, structural studies of ions in the gas phase have attracted significant attention as a means of studying intrinsic interactions of biomolecules in the absence of solvent to further understand fundamental aspects of protein folding. Examination of gas-phase structure/ conformations of protein ions has primarily been studied by ion mobility [4,5] and gas-phase hydrogendeuterium exchange [6][7][8][9][10] methods, with some recent studies [11,12] using the "gentle" dissociation process of electron capture dissociation (ECD) [13]. In addition, unfolding and folding transitions of gas-phase ions have been studied by exciting the ions via laser irradiation [8,11,12] or energetic collisions [5,8,9] followed by analysis of structure/conformation by the aforementioned methods.…”

mentioning

confidence: 99%

Evidence for unfolding and refolding of gas-phase cytochrome c ions in a Paul trap

Badman

Myung

Clemmer

2005

127

136

The folding pathways of gas-phase cytochrome c ions produced by electrospray ionization have been studied by an ion trapping/ion mobility technique that allows conformations to be examined over extended timescales (10 ms to 10 s). The results show that the ϩ9 charge state emerges from solution as a compact structure and then rapidly unfolds into several substantially more open structures, a transition that requires 30-60 ms; over substantially longer timescales (250 ms to 10 s) elongated states appear to refold into an array of folded structures. The new folded states are less compact than those that are apparent during the initial unfolding. Apparently, unfolding to highly open conformations is a key step that must occur before ϩ9 ions can sample more compact states that are stable at longer times. . Recently, structural studies of ions in the gas phase have attracted significant attention as a means of studying intrinsic interactions of biomolecules in the absence of solvent to further understand fundamental aspects of protein folding. Examination of gas-phase structure/ conformations of protein ions has primarily been studied by ion mobility [4, 5] and gas-phase hydrogendeuterium exchange [6-10] methods, with some recent studies [11,12] using the "gentle" dissociation process of electron capture dissociation (ECD) [13]. In addition, unfolding and folding transitions of gas-phase ions have been studied by exciting the ions via laser irradiation [8,11,12] or energetic collisions [5,8,9] followed by analysis of structure/conformation by the aforementioned methods. The advantages of studying gas-phase ions is that one is able to study not only "naked" ions' intrinsic physical and chemical properties, but also the stepwise solvation of these ions [14 -16].We have recently developed a new method for studying the time-dependent behavior of gas-phase protein ions. In this approach we store ions in a Paul geometry ion trap for variable amounts of time and follow the structural transitions by analyzing the conformations of electrosprayed ions by a combined ion mobility/time-of-flight analysis [17,18]. In this study we show evidence for structural transitions of the ϩ9