Disulfide bond cleavages observed in SORI-CID of three nonapeptides complexed with divalent transition-metal cations.

Tandem MS sequencing of peptides that contain a disulfide bond is often hampered when using a slow heating technique. We show that complexation of a transition-metal ion with a disulfide-bridge-containing nonapeptide yields very rich tandem mass spectra, including fragments that involve the cleavage of the disulfide bond up to 56% of the total product ion intensity. On the contrary, MS/MS of the corresponding protonated nonapeptides results predominantly in fragments from the region that is not involved in the disulfide bond. Eleven different combinations of three nonapeptides and three metal ions were measured using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) combined with sustained off-resonance irradiation collision induced dissociation (SORI-CID). All observed fragments are discussed with respect to four different types of product ions: neutral losses, b/y-fragmentation with and without the disulfide bond cleavage, and losses of internal amino acids without rupture of the disulfide bridge. Furthermore, it is shown that the observed complementary fragment pairs obtained from peptide-metal complexes can be used to determine the region of the binding site of the metal ion. This approach offers an efficient way to cleave disulfide-bridged structures using low energy MS/MS, which leads to increased sequence coverage and more confidence in peptide or protein assignments.

Combined infrared multiphoton dissociation and electron-capture dissociation using co-linear and overlapping beams in Fourier transform ion cyclotron resonance mass spectrometry.

A novel set-up for Fourier transform ion cyclotron resonance mass spectrometry (FTICR) is reported for simultaneous infrared multiphoton dissociation (IRMPD) and electron-capture dissociation (ECD). An unmodified electron gun ensures complete, on-axis overlap between the electron and the photon beams. The instrumentation, design and implementation of this novel approach are described. In this configuration the IR beam is directed into the ICR cell using a pneumatically actuated mirror inserted into the ion-optical path. Concept validation was made using different combinations of IRMPD and ECD irradiation events on two standard peptides. The ability to perform efficient IRMPD, ECD and especially simultaneous IRMPD and ECD using lower irradiation times is demonstrated. The increase in primary sequence coverage, with the combined IRMPD and ECD set-up, also increases the confidence in peptide and protein assignments.

Electron capture dissociation at low temperatures reveals selective dissociations.

Electron capture dissociation at 86 K of the linear peptide Substance P produced just two backbone fragments, whereas at room temperature eight backbone fragments were formed. Similarly, with the cyclic peptide gramicidin S, just one backbone fragment was formed at 86 K but five at room temperature. The observation that some backbone scissions are active and others inactive, when all involve NC(alpha) cleavages and have a high rate constant, indicates that the more specific fragments at low temperatures reflects the reduced conformation heterogeneity at low temperatures. This is supported by reduced or inactive hydrogen loss, a channel that has previously been shown to be affected by conformation. The conclusion that the ECD fragments are a snapshot of the conformational (intramolecular solvation shell) heterogeneity helps explain how the relative intensities of ECD fragments can be different on different instrument and highlights the common theme in methodologies used to increase sequence coverage, namely an increase in the conformational heterogeneity of the precursor ion population.