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. ollisional activation of ions takes place in all practical mass spectrometry measurements, either as a side effect of a residual gas pressure, or for the specific purpose of collisional ion cooling, focusing or collision induced dissociation. Modeling of the collisionally induced ion activation process in mass spectrometry has been the focus of extensive study [1][2][3][4][5]. Of particular importance is the collisional activation of ions in radio frequency (RF) ion traps and guides, where a substantial bath gas pressure and long residence times result in large numbers of the ionneutral collisions [6]. Several research groups have proposed using the effective temperature as a measure of ion activation under conditions of multiple ionneutral collisions [7][8][9][10][11][12]. As such, the effective temperature concept proved to be very useful in the interpretation of ion activation data obtained for various mass spectrometry experiments, in particular in quadrupole ion trap experiments [8,9,[13][14][15]. Generally, the internal energy of ions produced at high pressures (e.g., by the electrospray ionization process) can be characterized in terms of effective ion temperature [16 -20]. The effective temperature T eff of an ion, moving in a bath gas under the influence of electric fields, can be expressed through the ion drift velocity V drift as follows:2 , where T is the gas temperature, and C T is a model dependent proportionality coefficient [9,12,21]. This approximation is applicable to conditions when the force acting on an ion is constant during a time interval longer than the ion velocity relaxation time (i.e., the drift motion approximation) [22]. In the lower ...

In proteomics, effective methods are needed for identifying the relatively limited subset of proteins displaying significant changes in abundance between two samples. One way to accomplish this task is to target for identification by MS/MS only the "interesting" proteins based on the abundance ratio of isotopically labeled pairs of peptides. We have developed the software and hardware tools for online LC-FTICR MS/MS studies in which a set of initially unidentified peptides from a proteome analysis can be selected for identification based on their distinctive changes in abundance following a "perturbation". We report here the validation of this method using a mixture of standard proteins combined in different ratios after isotopic labeling. We also demonstrate the application of this method to the identification of Shewanella oneidensis peptides/proteins exhibiting differential abundance in sub-oxic vs. aerobic cell cultures.

Bottom-up proteomics (analyzing peptides that result from protein digestion) has demonstrated capability for broad proteome coverage and good throughput. However, due to incomplete sequence coverage, this approach is not ideally suited to the study of modified proteins. The modification complement of a protein can best be elucidated by analyzing the intact protein. 2-DE, typically coupled with the analysis of peptides that result from in-gel digestion, is the most frequently applied protein separation technique in MS-based proteomics. As an alternative, numerous column-based liquid phase techniques, which are generally more amenable to automation, are being investigated. In this work, the combination of size-exclusion chromatography (SEC) fractionation with RPLC-Fourier-transform ion cyclotron resonance (FTICR)-MS is compared with the combination of RPLC fractionation with CIEF-FTICR-MS for the analysis of the Shewanella oneidensis proteome. SEC-RPLC-FTICR-MS allowed the detection of 297 proteins, as opposed to 166 using RPLC-CIEF-FTICR-MS, indicating that approaches based on LC-MS provide better coverage. However, there were significant differences in the sets of proteins detected and both approaches provide a basis for accurately quantifying changes in protein and modified protein abundances.

We report on the use of a jet disrupter electrode in an electrodynamic ion funnel as an electronic valve to regulate the intensity of the ion beam transmitted through the interface of a mass spectrometer in order to perform automatic gain control (AGC). The ion flux is determined by either directly detecting the ion current on the conductance limiting orifice of the ion funnel or using a short mass spectrometry acquisition. Based upon the ion flux intensity, the voltage of the jet disrupter is adjusted to alter the transmission efficiency of the ion funnel to provide a desired ion population to the mass analyzer. Ion beam regulation by an ion funnel is shown to provide control to within a few percent of a targeted ion intensity or abundance. The utility of ion funnel AGC was evaluated using a protein tryptic digest analyzed with liquid chromatography Fourier transform ion cyclotron resonance (LC-FTICR) mass spectrometry. The ion population in the ICR cell was accurately controlled to selected levels, which improved data quality and provided better mass measurement accuracy. M ass spectrometry (MS) has become a vital tool in biological research. This information-rich detection method can produce sensitive, qualitative, and quantitative measurements, and provides the basis for characterizing proteins, identifying novel biomarkers, and studying protein interactions within biological networks and pathways. Many challenges in protein analysis stem from sample complexity, e.g., a typical mammalian cell can have protein abundances ranging from less than a few hundred to tens of millions of copies [1]. A focus in MS research continues to be the development of techniques to better handle the broad range of relative abundances from a single sample. Developments have included chemical methods [2][3][4], coupling MS to separation techniques [5,6], and improvements in instrumentation [7][8][9]. An example of the latter is automatic gain control (AGC) [8 -11], first developed by the Finnigan Corporation (now Thermo Electron Corporation) [9]. AGC provides automated regulation to a dynamic ion flux transmitted from the source of the instrument (common in liquid chromatography {LC} coupled MS experiments), resulting in a more constant ion population in the mass analyzer. AGC accomplishes this regulation by monitoring the ion production from the ion source (typically with a pre-scan) and providing on-the-fly adjustments of the ion accumulation time of an ion trap.The regulation or control of the ion population is important to the operation of most mass spectrometers, and particularly those based upon ion trapping where performance is degraded by excessive space charge. For example, a key source of mass error in Fourier transform ion cyclotron resonance (FTICR) MS comes from the effect of excessive space charge [12][13][14]. Linear and 3-D ion traps also experience detrimental effects from excessive space charge. These space charge effects lead to shifts in secular frequencies, changes in optimal excitation amplitude, and plasma e...