Tandem mass spectrometry in an electrostatic linear ion trap modified for surface-induced dissociation.

A variety of ion traps are used in mass spectrometry. A key feature shared by most of them is the ability to perform tandem mass spectrometry (MS/MS). The Orbitrap is perhaps the most notable ion trap in which MS/MS has yet to be performed. An electrostatic linear ion trap (ELIT) is analogous to an orbitrap in that ions are trapped using solely electrostatic fields. However, the relatively simple ion motion within an ELIT facilitates analysis of fragment ions produced within the device. In this report, we describe an ELIT to which we have added a target for surface induced dissociation (SID). When combined with our previously described method for isolating a precursor ion trapped in an ELIT,1 this apparatus enables MS/MS to be performed. Measurement of product ion m/z is facilitated by the fact that the ELIT is isochronous over the energy range of 1850-2000 eV so that changes to ion energy during SID do not cause major m/z shifts. We demonstrate MS/MS by isolating and dissociating each compound in a four component mixture of tetraalkylphosphonium cations. We also discuss the optimization of collision energy and the length of time that the SID target is available for collision, two parameters that are important in the performance of these experiments.

Absorption mode Fourier transform electrostatic linear ion trap mass spectrometry.

In Fourier transform mass spectrometry, it is well-known that plotting the spectrum in absorption mode rather than magnitude mode has several advantages. However, magnitude spectra remain commonplace due to difficulties associated with determining the phase of each frequency at the onset of data acquisition, which is required for generating absorption spectra. The phasing problem for electrostatic traps is much simpler than for Fourier transform ion cyclotron resonance (FTICR) instruments, which greatly simplifies the generation of absorption spectra. Here, we present a simple method for generating absorption spectra from a Fourier transform electrostatic linear ion trap mass spectrometer. The method involves time shifting the data prior to Fourier transformation in order to synchronize the onset of data acquisition with the moment of ion acceleration into the electrostatic trap. Under these conditions, the initial phase of each frequency at the onset of data acquisition is zero. We demonstrate that absorption mode provides a 1.7-fold increase in resolution (full width at half maximum, fwhm) as well as reduced peak tailing. We also discuss methodology that may be applied to unsynchronized data in order to determine the time shift required to generate an absorption spectrum.

Nondestructive tandem mass spectrometry using a linear quadrupole ion trap coupled to a linear electrostatic ion trap.

A novel hybrid tandem mass spectrometer is presented that combines a linear quadrupole ion trap (QLIT) with a linear electrostatic ion trap (ELIT), which is composed of opposing ion mirrors. The QLIT is used both as an accumulation device for the pulsed injection of ions into the ELIT and as a collision cell for ions released from the ELIT and back into the QLIT. Ions are subjected to mass analysis in the ELIT via Fourier transformation of the time-domain signal obtained from an image current measurement using a pick-up electrode in the field-free region of the ELIT. The nondestructive nature of ion detection and the relatively straightforward axial entrance and exit of ions into and from the ELIT allow for the execution of nondestructive tandem mass spectrometry experiments whereby both the initial mass spectrum and the product ion spectrum are obtained on the same initial ion population. The timed pulsing of a deflection electrode, in conjunction with the release of ions from the ELIT, allows for the selection of precursor ions for recapture by the QLIT. The transfer of ions back and forth between the QLIT and ELIT is illustrated with Cs ions, the selection of precursor ions is demonstrated with isotopes of tetraoctylammonium cations, and complete nondestructive tandem mass spectrometry experiments are demonstrated with a mixture of angiotensin II and bradykinin cations. With the current apparatus, the efficiency for the process of recapturing ions and then reinjecting them into the ELIT is 35%-40%. The instrument is capable of isolating an ion from a neighbor with a mass as close as 1 part in 500, with negligible loss of the desired species.

Adaptation of a 3-D quadrupole ion trap for dipolar DC collisional activation.

Means to allow for the application of a dipolar DC pulse to the end-cap electrodes of a three-dimensional (3-D) quadrupole ion trap for as short as a millisecond to as long as hundreds of milliseconds are described. The implementation of dipolar DC does not compromise the ability to apply AC waveforms to the end-cap electrodes at other times in the experiment. Dipolar DC provides a nonresonant means for ion acceleration by displacing ions from the center of the ion trap where they experience stronger rf electric fields, which increases the extent of micro-motion. The evolution of the product ion spectrum to higher generation products with time, as shown using protonated leucine enkephalin as a model protonated peptide, illustrates the broad-band nature of the activation. Dipolar DC activation is also shown to be effective as an ion heating approach in mimicking high amplitude short time excitation (HASTE)/pulsed Q dissociation (PQD) resonance excitation experiments that are intended to enhance the likelihood for observing low m/z products in ion trap tandem mass spectrometry.

Saturation transfer double-difference NMR spectroscopy using a dual solenoid microcoil difference probe.

An experiment designed to collect a saturation transfer double difference (STDD) NMR spectrum using a solenoid microcoil NMR difference probe is reported. STDD-NMR allows the investigation of ligand-biomolecule binding, with moderate concentration requirements for unlabeled molecular targets and the ability to discern binding events in the presence of non-binding ligands. The NMR difference probe acquires the signals from two different samples at once, and cancels common signals automatically through a mechanism of switching between parallel excitation and serial acquisition of the sample signals. STDD spectra were acquired on a system consisting of human serum albumin and two ligands, octanoic acid and glucose. The non-binding ligand, glucose, was cancelled internally through phase cycling, while the protein signal was subtracted automatically by the difference probe. The proton NMR resonance signal from octanoic acid remained in the double difference spectrum. This work demonstrates that the double difference can be performed both internally and automatically through the utilization of the solenoid microcoil NMR difference probe and STDD-NMR pulse sequence, resulting in a clean signal from the binding ligand with good protein background subtraction and an overall favorable result when compared to the conventional approach.

Results with an apparatus for pressurized planar electrochromatography.

Pressurized planar electrochromatography (PPEC) is a fast and efficient planar chromatographic technique. The mobile phase is driven by electroosmotic flow, while the system is pressurized in a manner that allows heat to flow between the sorbent layer and the pressurizing medium. The reproducibility of solute retention was not satisfactory in the initial report describing PPEC. In the current report, this reproducibility is improved by better control of several experimental variables. The pressure at which PPEC is performed is now free of drift, and the temperature at which the layer is preconditioned is maintained to within +/-1 degrees C. The best reproducibility of retention is obtained when the plate is soaked in the mobile phase for a defined time before each run. In the original prototype, the temperature of the sorbent layer was not controlled. In the present apparatus, water, at a constant temperature between 3 and 60 degrees C, is circulated through channels in the two die blocks that pressurize the layer. The highest efficiency is obtained at an intermediate temperature. This behavior is ascribed to high resistance to mass transfer at the lower temperatures and increased diffusion at higher temperatures. Efficiency, as measured by the number of theoretical plates, increases with increasing migration distance. The height equivalent of a theoretical plate diminishes with increasing migration distance, and values as low as 0.0106 mm are obtained under appropriate conditions. This extrapolates to 94 000 plates/m. Manual spotting was used in this report. Evidence is presented that substantially better efficiency would be obtained if the initial spot size were smaller. The efficiency of PPEC in its current form is illustrated by a chromatogram showing the separation of nine solutes in 2 min. PPEC was also performed with TLC plates in a back-to-back configuration, and this doubles the number of samples that can be simultaneously separated.

NMR difference spectroscopy with a dual saddle-coil difference probe.

A new difference probe for nuclear magnetic resonance (NMR) spectroscopy is presented. The difference probe uses two saddle-shaped coils to excite and detect two samples simultaneously. The samples are held in a specially modified 3-mm NMR tube with an Ultem plastic disk to separate the samples. The probe's resonant circuit contains two crossed diodes that passively switch the relative phase of each coil during the NMR experiment. The result is a difference spectrum from the two samples. The degree of cancellation of common signals was determined to be approximately 90%, and the application of the probe to relaxation-edited difference spectroscopy for identifying protein-ligand interactions was demonstrated using glutathione and glutathione S-transferase binding protein.

Apparatus and initial results for pressurized planar electrochromatography.

Pressurized planar electrochromatography (PPEC) is a new planar chromatographic technique in which the mobile phase is driven by electroosmotic flow, while the sorbent layer is pressurized in a manner that allows heat to flow from the layer through an electrically insulating, thermally conducting, sheet of aluminum nitride ceramic. A prototype apparatus for performing PPEC is described. Separation by PPEC is faster than by conventional TLC, and an example is presented of a 24-fold enhancement in the speed of separation. PPEC was performed on both regular and high-performance C18 layers, and the latter yield substantially faster separation. The sorbent layer requires conditioning at elevated temperature before use, and solute migration velocity increases with this temperature. The flow rate increases in a linear manner with increasing voltage and diminishes in a nonlinear manner with increasing pressure. Both electrical current and Joule heating diminish with increasing pressure, and the diminution of flow at high pressure can be compensated by an increase in voltage. PPEC is more efficient than classical TLC. Theoretical plate heights diminish with increasing Rf and are in the range 29-21 and 55-27 microm for the high-performance and regular plates, respectively. PPEC retains the advantages of classical TLC but has the ability to separate a substantially higher number of samples simultaneously. An example is presented on the separation of nine samples in 1 min on a 2.5 cm x 10 cm sorbent layer.

NMR difference probe: a dual-coil probe for NMR difference spectroscopy.

A unique probe designed to acquire nuclear magnetic resonance difference spectra of two samples is presented. The NMR Difference Probe contains two sample coils in a resonant circuit that switches between parallel excitation and serial acquisition to cancel common signals such as solvent peaks and impurities. Two samples containing a common analyte, acetonitrile, were used to demonstrate signal cancellation in a difference spectrum collected with a single pulse experiment. The cancellation was over 96% effective. The approach described has applications in the areas of solvent subtraction and spectral simplification.