Collisional activation of ions in RF ion traps and ion guides: The effective ion temperature treatment

Ion transfer and storage using inhomogeneous radio frequency (RF) electric fields in combination with gas-assisted ion cooling and focusing constitutes one of the basic techniques in mass spectrometry today. The RF motion of ions in the bath gas environment involves a large number of ion-neutral collisions that leads to the internal activation of ions and their effective "heating" (when a thermal distribution of internal energies results). The degree of ion activation required in various applications may range from a minimum level (e.g., slightly raising the average internal energy) to an intense level resulting in ion fragmentation. Several research groups proposed using the effective temperature as a measure of ion activation under conditions of multiple ion-neutral collisions. Here we present approximate relationships for the effective ion temperature relevant to typical operation modes of RF multipole devices. We show that RF ion activation results in near-thermal energies for ions occupying an equilibrium position at the center of an RF trap, whereas increased ion activation can be produced by shifting ions off-center, e.g., by means of an external DC electric field. The ion dissociation in the linear quadrupole ion trap using the dipolar DC ion activation has been observed experimentally and interpreted in terms of the effective ion temperature.     

Gene expression profiling using advanced mass spectrometric approaches

In the era of systems biology, computational and high-throughput experimental biological approaches are increasingly being combined to provide global snapshots of entire genomes and proteomes under tissue- and disease-specific conditions. The aim is to identify proteins changing in concentration and/or post-translational state and/or location, and develop a better molecular level understanding of the operation of biological systems. Here we describe an approach for comparative proteomics that builds upon the combination of high-performance nano-scale separations with the high-mass measurement accuracy, mass-resolving power and sensitivity of Fourier transform ion cyclotron resonance mass spectrometry to provide broad dynamic range, comprehensive and quantitative proteome measurements.     

A dynamic ion cooling technique for FTICR mass spectrometry

A fast dynamic ion cooling technique based upon the adiabatic invariant phenomenon for Fourier transform ion cyclotron resonance mass spectrometry (FTICR) is presented. The method cools ions in the FTICR trap more efficiently, within a few hundred milliseconds without the use of a buffer gas, and results in a substantial signal enhancement. All performance aspects of the FTICR spectrum, e.g., peak intensities, mass resolution, and mass accuracy, improve significantly compared with cooling based on ion-ion interactions. The method may be useful in biological applications of FTICR, such as in proteomic studies involving extended on-line liquid chromatography (LC) separations, in which both the duty cycle and mass accuracy are crucially important.     

Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry.

The patterns of gene expression, post-translational modifications, protein/biomolecular interactions, and how these may be affected by changes in the environment, cannot be accurately predicted from DNA sequences. Approaches for proteome characterization are generally based upon mass spectrometric analysis of in-gel digested two dimensional polyacrylamide gel electrophoresis (2-D PAGE) separated proteins, allowing relatively rapid protein identification compared to conventional approaches. This technique, however, is constrained by the speed of the 2-D PAGE separations, the sensitivity limits intrinsic to staining necessary for protein visualization, the speed and sensitivity of subsequent mass spectrometric analyses for identification, and the limited ability for accurate quantitative measurements based on differences in spot intensity. We are presently developing alternative approaches for proteomics based upon the combination of fast capillary electrophoresis, or other suitable chromatographic separations, and the high mass accuracy and sensitivity obtainable with unique Fourier transform ion cyclotron resonance (FTICR) mass spectrometers available at our laboratory. Several approaches are presently being pursued; one based upon the analysis of intact proteins and the second upon approaches for global protein digestion and accurate peptide mass analysis. Quantitation of protein/peptide levels are based on using two or more stable-isotope labeled versions of proteomes which are combined to obtain precise quantitation of relative protein abundances. We describe the status of our efforts towards the development of a high-throughput proteomics capability and present initial results for application to several microorganisms and discuss our efforts for extending the developed capability to mammalian proteomes.     

Influence of mass resolution on species matching in accurate mass and retention time (AMT) tag proteomics experiments

Diverse mass spectrometric instruments have been used to provide data for accurate mass and retention time (AMT) tag proteomics analyses, including ion trap, quadrupole time-of-flight, and Fourier transform mass spectrometry (FTMS). An important attribute of these instruments, beside mass accuracy, is their spectral resolution. In fact, the ability to separate peaks with close m/z values is likely to play a major role in enabling species identification and matching in analyses of very complex proteomics samples. In FTMS, resolution is directly proportional to the detection period and can therefore be easily tuned. We took advantage of this feature to investigate the effect of resolution on species identification and matching in an AMT tag experiment. Using an Arabidopsis thaliana chloroplast protein extract as prototypical 'real-life' sample, we have compared the number of detected features, the optimal mass tolerance for species matching, the number of matched species and the false discovery rate obtained at various resolution settings. It appears that while the total number of matches is not significantly affected by a reduction of resolution in the range investigated, the confidence level of identifications significantly drops as evidenced by the estimated false discovery rate.     

Tailored noise waveform/collision-induced dissociation of ions stored in a linear ion trap combined with liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry

A new collision-induced dissociation (CID) technique based on broadband tailored noise waveform (TNW) excitation of ions stored in a linear ion trap has been developed. In comparison with the conventional sustained off-resonance irradiation (SORI) CID method commonly used in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), this MS/MS technique increases throughput by eliminating the long pump-down delay associated with gas introduction into the high vacuum ICR cell region. In addition, the TNW-CID method speeds spectrum acquisition since it does not require Fourier transformation, calculation of resonant frequencies and generation of the excitation waveforms. We demonstrate TNW-CID coupled with on-line capillary reverse-phase liquid chromatography separations for the identification of peptides. The experimental results are compared with data obtained using conventional quadrupole ion trap MS/MS and SORI-CID MS/MS in an ICR cell.     

ESI-FTICR mass spectrometry employing Data-dependent external ion selection and accumulation

Data-dependent external m/z selection and accumulation of ions is demonstrated in use with ESI-FTICR instrumentation, with two different methods for ion selection being explored. One method uses RF/DC quadrupole filtering and is described in use with an 11.5 tesla (T) FTICR instrument, while the second method employs RF-only resonance dipolar excitation selection and is described in use with a 3.5 T FTICR instrument. In both methods ions are data-dependently selected on the fly in a linear quadrupole ion guide, then accumulated in a second linear RF-only quadrupole trap that immediately follows. A major benefit of ion preselection prior to external accumulation is the enhancement of ion populations for low-level species. This development is expected to expand the dynamic range and sensitivity of FTICR for applications including analysis of complex polypeptide mixtures (e.g., proteomics).     

UTILIZATION OF TOOLS FOR SUCCESSFUL. TROUBLESHOOTING OF STRUCTURAL PROBLEMS

A structural failure problem was solved using an integrated and iterative program of testing and analysis. The steps taken in solving the problem were: analytical calculations; operational testing; qualifications of analytical results; problem identification; design of corrective action; and confirmatory testing.