Higher Order Structure in the Gas Phase Reflects Solution Structure

Abstract

As new methods for volatilizing and ionizing biological molecules for mass spectrometry (MS) analysis are developed, the conformation of gas-phase biomolecule ions and how it relates to solution-phase reactivity and structure has been generating considerable interest. 1 The conformations of gas-phase ions of large polypeptides formed by electrospray ionization (ESI) have been probed by a variety of means, including ion-molecule reactions, 2 collision cross-section measurements, 3 and hydrogendeuterium-exchange (H/D). 4 The number of exchangeable protons reflects the openness of the protein conformation. Ion mobility measurements have resolved gas-phase structural isomers for a number of proteins. 5 The charge state distribution produced by ESI as a function of pH, 6 solvent content, 7 and metal-binding properties 8 has also been related to the protein conformation in solution. Another recent application of ESI-MS includes the study of noncovalently bound complexes, 9,10 for which MS has unique advantages in determining complex stoichiometry of the binding partners. Characteristics of the native solution state of proteins and oligonucleotides and their interactions with other biomolecules can be also captured by MS measurements.Tandem mass spectrometry (MS/MS) with collisionally activated dissociation (CAD) has been used also to explore the reactivities of large ions. Mass-analyzed kinetic energy (MIKE) spectrometry was used to study the stability of secondary structure in the absence of solvent for the peptide melittin. 11 The CAD mass spectra of peptide-metal ion complexes show that relatively selective bond cleavages can be induced, as specific complexation with metal ions can direct specific dissociation pathways. 12 Smith and co-workers' CAD study of 12 kDa cytochrome c proteins suggests that the fragmentation pattern could be influenced by the protein's higher order structure. 13 However, from these studies, it is not clear whether these three-dimensional structures in a solvent-less environment bear any resemblance to the conformation in the solution phase. We present an example in which the gas-phase MS/MS-based measurements of a polypeptide are consistent with its known solution-phase structure.RES-701-1 (I), isolated from Streptomyces sp. RE-701, is a potent and selective endothelin ET B receptor antagonist. 14 The hexadecapeptide possesses a unique linkage between the -carbonyl carbon of Asp-9 and the R-amino group of Gly-1, forming a 9-residue cyclic "head" structure and a 7-residue "tail" (see Scheme 1). However, a synthetic version of the peptide (II) is characterized by an affinity for the ET B receptor reduced by nearly 3 orders of magnitude (IC 50 of 10 nM for the natural peptide and 6.5 µM for the synthetic peptide). 15,16 High-resolution, exact mass measurements with ESI show the two peptides to be within (0.004 mass units ((2 ppm) of the expected value (measured monoisotopic mass of I was 2041.8520, and a mass of 2041.8492 for II; expected mass from sequence is 2041.8536). 17 Dramatic differ...

The pH Dependence of Product Ion Spectra Obtained from Precursor Ions with the Same Charge Number in ESI of Carbonic Anhydrase 2

“…21) In contrast, our finding is not applicable to proteins whose conformation is stabilized by strong intramolecular interactions. 22,23) However, the findings in these prior reports as well as our present study indicated that the product ion spectrum provides an adequate description of the folding state of a protein.…”

Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

“…Many common product ions were detected for both topoisomers, but with different 2+ , more intense for the lasso topoisomer, correspond to lasso-structured product ions, maintained through R11 and R15 side chains located above and below the ring. Thus, as for MccJ25, the ergodic gas-phase fragmentations of capistruin appear to result in two dissociative processes, (1) the former preserving the lasso structure and yielding the [b 15 -H 2 O] 2+ to [b 18 -H 2 O] 2+ product ions, and (2) the latter yielding y/b series corresponding to cleavages in the Cterminal tail, which indicates a previous precursor ion isomerization through opening of the macrolactam ring. However, the short sequence of the loop did not permit to generate two-peptide product ions, which require two bond cleavages in the loop region.…”

“…Cleavage within the Y9-I18 region, either in solution by enzymatic cleavage or acidic hydrolysis, or in the gas phase upon collision-induced dissociation (CID), generates two peptide complexes associated through the steric hindrance provided by the side chains of F19 and Y20 [15][16][17]. A particular gas-phase fragmentation pattern upon CID, compared with the corresponding synthetic peptide encompassing a macrolactam ring, has also been reported for the lasso peptide RES 701-1 [18], but generally only NMR could unambiguously characterize the threading of the C-terminal tail into the macrolactam rings [7,8,19]. Capistruin is a lasso peptide secreted by Burkholderia thailandensis E264 discovered by genome mining, which exhibits antibacterial activities against closely related Burkholderia and Pseudomonas species [8].…”

Dissociation of different conformations of ubiquitin ions

“…In addition to chemical probes of gas phase ion structure [1][2][3][4][5][6][7][8][9][10][11][12][13][14], a number physical methods are being developed to examine gas-phase peptide and protein ion structure [15][16][17][18][19][20][21][22][23][24][25][26][27], including collisional activation to examine fragmentation pathways [18 -23, 25-27]. A few studies have suggested that ion conformation may influence fragmentation pathways [19,23,27,28]. In this paper, we examine this issue for the fragmentation of a series of compact and elongated conformations of ubiquitin ions.…”