The Mass Spectrometry of Helical Unfolding in Peptides

Abstract

Two model peptides, melittin and a growth hormone releasing factor (GRF) analog, have been studied by mass spectrometry and tandem mass spectrometry during the course of their deuterium exchange. Both peptides are known from previous work to form α-helices in solution. When the peptides are exposed to deuterated solvents, their masses increase as deuterium atoms replace protons in the exchangeable sites of the peptides. The mass spectrometry results clearly indicate multiple populations of exchangeable protons: Some exchange very fast, and are presumably on the surface and not involved in hydrogen bonding; others exchange much more slowly, indicating that they are probably participating in hydrogen bonding.Tandem mass spectrometric experiments were conducted, and the masses of the product (fragment) ions were used to determine where in the peptide the deuterium atoms were incorporated. The results agree very well with NMR studies of the same peptides. Melittin appears as two helical segments with a kink around Pro-14. The GRF analog contains a single long helix, spanning almost the entire length of the peptide. The dynamics of the unfolding of the helices can also be explored by observing how the exchange progresses with time.

Hydrogen Exchange-Mass Spectrometry

“…The actual gain in spatial resolution depends on the size of the peptides and is often in the range of 5-10 residues. Higher spatial resolution has been obtained by using multiple proteases (27), differences in overlapping fragments (29), and collision-induced dissociation (MS/MS) (10,35,36). The success of all of these approaches depends on being able to digest proteins under conditions where isotope exchange is slow.…”

Electrospray ionization mass spectrometry as a tool to analyze hydrogen/deuterium exchange kinetics of transmembrane peptides in lipid bilayers

“…It is known that proton mobility within smaller protonated gas-phase peptide ions can often be sufficiently rapid to scramble deuterium labeling (28). However, recent findings of Anderegg et al (17) and Deng et al (18) have shown that under certain conditions, hydrogen exchange at individual peptide amide linkages can be determined by collision-induced dissociation MS. The results obtained in the present study are fully consistent with the expected exchange properties of the peptides based on the model in Fig.…”

Collisional Activation by MALDI Tandem Time-of-flight Mass Spectrometry Induces Intramolecular Migration of Amide Hydrogens in Protonated Peptides

“…For a first hand view, gas-phase fragmentation in the mass spectrometer may appear to be the logical choice for an auxiliary method providing the desired site-specific informa-tion. Accordingly it has been widely adopted in the attempt to identify specific sites that have become deuterated in solution 1 H/ 2 H exchange experiments (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). It is, however, mandatory for this approach that the level of intramolecular hydrogen ( 1 H/ 2 H) migration upon ion activation is negligible.…”