Spectroscopic Evidence for an Oxazolone Structure in Anionic b-Type Peptide Fragments

Infrared spectra of anionic b-type fragments generated by collision induced dissociation (CID) from deprotonated peptides are reported. Spectra of the b2 fragments of deprotonated AlaAlaAla and AlaTyrAla have been recorded over the 800–1800 cm–1 spectral range by multiple-photon dissociation (MPD) spectroscopy using an FTICR mass spectrometer in combination with the free electron laser FELIX. Structural characterization of the b-type fragments is accomplished by comparison with density functional theory calculated spectra at the B3LYP/6-31++G(d,p) level for different isomeric structures. Although diketopiperazine structures represent the energetically lowest isomers, the IR spectra suggest an oxazolone structure for the b2 fragments of both peptides. Deprotonation is shown to occur on the oxazolone α-carbon, which leads to a conjugated structure in which the negative charge is practically delocalized over the entire oxazolone ring, providing enhanced gas-phase stability

Mass-selective soft-landing of protein assemblies with controlled landing energies.

Selection and soft-landing of bionanoparticles in vacuum is potentially a preparative approach to separate heterogeneous mixtures for high-resolution structural study or to deposit homogeneous materials for nanotechnological applications. Soft-landing of intact protein assemblies however remains challenging, due to the difficulties of manipulating these heavy species in mass-selective devices and retaining their structure during the experiment. We have developed a tandem mass spectrometer with the capability for controlled ion soft-landing and ex situ visualization of the soft-landed particles by means of transmission electron microscopy. The deposition conditions can be controlled by adjusting the kinetic energies of the ions by applying accelerating or decelerating voltages to a set of ion-steering optics. To validate this approach, we have examined two cage-like protein complexes, GroEL and ferritin, and studied the effect of soft-landing conditions on the method's throughput and the preservation of protein structure. Separation, based on mass-to-charge ratio, of holo- and apo-ferritin complexes after electrospray ionization enabled us to soft-land independently the separated complexes on a grid suitable for downstream transmission electron microscopy analysis. Following negative staining, images of the soft-landed complexes reveal that their structural integrity is largely conserved, with the characteristic central cavity of apoferritin, and iron core of holoferritin, surviving the phase transition from liquid to gas, soft-landing, and dehydration in vacuum

Mass-selective soft-landing of protein assemblies with controlled landing energies.

Selection and soft-landing of bionanoparticles in vacuum is potentially a preparative approach to separate heterogeneous mixtures for high-resolution structural study or to deposit homogeneous materials for nanotechnological applications. Soft-landing of intact protein assemblies however remains challenging, due to the difficulties of manipulating these heavy species in mass-selective devices and retaining their structure during the experiment. We have developed a tandem mass spectrometer with the capability for controlled ion soft-landing and ex situ visualization of the soft-landed particles by means of transmission electron microscopy. The deposition conditions can be controlled by adjusting the kinetic energies of the ions by applying accelerating or decelerating voltages to a set of ion-steering optics. To validate this approach, we have examined two cage-like protein complexes, GroEL and ferritin, and studied the effect of soft-landing conditions on the method's throughput and the preservation of protein structure. Separation, based on mass-to-charge ratio, of holo- and apo-ferritin complexes after electrospray ionization enabled us to soft-land independently the separated complexes on a grid suitable for downstream transmission electron microscopy analysis. Following negative staining, images of the soft-landed complexes reveal that their structural integrity is largely conserved, with the characteristic central cavity of apoferritin, and iron core of holoferritin, surviving the phase transition from liquid to gas, soft-landing, and dehydration in vacuum

A modular data and control system to improve sensitivity, selectivity, speed of analysis, ease of use, and transient duration in an external source FTICR-MS

We present here a new Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) controller designed and constructed to meet the growing need for increased speed, memory, and ease of use. The system realizes these goals via the first published application of fast PXI bus technology and by employing a graphical user interface (GUI) for control of all aspects of ion production, delivery, containment, manipulation of internal and kinetic energies, and measurement in an external source instrument. Additionally, new hardware for monitoring and control of these aspects and for processing extended datasets both to and from the instrument have been implemented.\u3cbr/\u3e\u3cbr/\u3eThe modular nature of the control hardware makes the instrument platform, in this case a modified external source 7T FTICR-MS, irrelevant. The GUI consists of two separate modules; one provides a temporal representation of the pulses, voltages (rf and dc), and dc heater currents that control all aspects of the experiment while the other provides complex data analysis capabilities and design of excitation waveforms. Real-time monitoring of the transient signal is available in this module as well as near real-time monitoring of the resulting mass spectrum (using a truncated dataset). A PXI bus with 40 digital to analog converters (DAC) and 64 digital (TTL) sources drive the source, optics, and trapping functions of the instrument as well as the other peripheral hardware. Acquisition is realized using a VME bus with a TTL triggered program resident on an embedded processor to minimize dead time. The acquisition system is equipped with 192 MB memory for both excitation and detection waveforms with FIFO buffering to provide full rate bandwidth of 10 MHz, and four digital down converters (DDCs) to enable mixing of heterodyne signals for narrow band measurements completes the ensemble.\u3cbr/\u3

Spectroscopic Identification of Cyclic Imide b(2)-Ions from Peptides Containing Gln and Asn Residues

Contains fulltext : 117239pos.pdf (postprint version ) (Open Access

An external matrix assisted laser desorption ionization source for flexible FT-ICR mass spectrometry imaging with internal calibration on adjacent samples

We describe the construction and application of a new MALDI source for FT-ICR mass spectrometry imaging. The source includes a translational X-Y positioning stage with a 10×10 cm range of motion for analysis of large sample areas, a quadrupole for mass selection, and an external octopole ion trap with electrodes for the application of an axial potential gradient for controlled ion ejection. An off-line LC MALDI MS/MS run demonstrates the utility of the new source for data- and position-dependent experiments. A FT-ICR MS imaging experiment of a coronal rat brain section yields ∼200 unique peaks from m/z 400-1100 with corresponding mass-selected images. Mass spectra from every pixel are internally calibrated with respect to polymer calibrants collected from an adjacent slide