John E. P. Syka

A two-dimensional quadrupole ion trap mass spectrometer

The use of a linear or two-dimensional (2-D) quadrupole ion trap as a high performance mass spectrometer is demonstrated. Mass analysis is performed by ejecting ions out a slot in one of the rods using the mass selective instability mode of operation. Resonance ejection and excitation are utilized to enhance mass analysis and to allow isolation and activation of ions for MSn capability. Improved trapping efficiency and increased ion capacity are observed relative to a three-dimensional (3-D) ion trap with similar mass range. Mass resolution comparable to 3-D traps is readily achieved, including high resolution at slower scan rates, although adequate mechanical tolerance of the trap structure is a requirement. Additional advantages of 2-D over 3-D ion traps are also discussed and demonstrated.

Recent improvements in and analytical applications of advanced ion trap technology

Abstract A new method of ion trap operation is disclosed in which the trap functions in a mass selective instability mode. Relatively high pressures of a light gas are used to damp the ionic motion and increases in resolution, sensitivity and detection limit have been found. The use of an ion trap as a sensitive, compound-specific gas chromatographic detector is discussed.

Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry

We present a strategy for the analysis of the yeast phosphoproteome that uses endo-Lys C as the proteolytic enzyme, immobilized metal affinity chromatography for phosphopeptide enrichment, a 90-min nanoflow-HPLC/electrospray-ionization MS/MS experiment for phosphopeptide fractionation and detection, gas phase ion/ion chemistry, electron transfer dissociation for peptide fragmentation, and the Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation sites. From a 30-μg (≈600 pmol) sample of total yeast protein, we identify 1,252 phosphorylation sites on 629 proteins. Identified phosphoproteins have expression levels that range from <50 to 1,200,000 copies per cell and are encoded by genes involved in a wide variety of cellular processes. We identify a consensus site that likely represents a motif for one or more uncharacterized kinases and show that yeast kinases, themselves, contain a disproportionately large number of phosphorylation sites. Detection of a pHis containing peptide from the yeast protein, Cdc10, suggests an unexpected role for histidine phosphorylation in septin biology. From diverse functional genomics data, we show that phosphoproteins have a higher number of interactions than an average protein and interact with each other more than with a random protein. They are also likely to be conserved across large evolutionary distances.

Operation of a quadrupole ion trap mass spectrometer to achieve high mass/charge ratios

Abstract One of the principal limitations of the commercial quadrupole ion trap mass spectrometer is the relatively low limiting value of mass/charge ratio (650 Da per charge). This constraint limits applications of desorption ionization techniques which can produce ions of many thousand Da per charge. Several techniques for extending the mass/charge range of the quadrupole ion trap are presented. These include (i) the use of smaller electrodes, (ii) operation at lower radio frequencies, and (iii) resonance ion ejection using a voltage of appropriate frequency, applied symmetrically across the end-cap electrodes during the mass scan. The performance of each of these methods is compared using external ionization of alkali halide salts with Cs+ bombardment. Special attention is given to the effects of scan rate on resolution and a method of reducing the scan rate without loss of data is described. Mass measurement accuracy is studied in some detail, the mass shifts which occur using resonance ejection are characterized and this information is used to correct mass assignments. The relative merits of the three methods of mass range extension are assessed. Field inhomogeneities in the particular smaller electrodes used here apparently cause some loss of performance in these devices making them the least successful of the methods. Frequency reduction gives excellent results over a limited range of m/z values. However, resonance ejection can be used alone to achieve an even greater m/z extension and is the (single) method of choice. A combination of modest size and frequency reduction with axial modulation is probably an ideal solution for high mass biological mass spectrometry. Performance at high mass is illustrated by recording data on CsI clusters and on peptides. These experiments show that mass/charge measurements are accurate to better than 0.1% without external calibration, that a mass resolution of 3000 FWHM is achieved (unit resolution at 50% valley to 3000), and that a mass/charge range in excess of 70 000 Da per charge is accessible.

High resolution on a quadrupole ion trap mass spectrometer

By using a modified ion trap mass spectrometer, resolution in excess of 30,000 (FWHM) at m I z 502 is demonstrated. The method of increasing resolution in the ion trap mass spectrometer operated in the mass-selective instability mode depends on decreasing the rate of scanning the primary radio frequency amplitude as well as using resonance ejection at the appropriate frequency and amplitude. A theoretical basis for the method is introduced.

Tandem mass spectrometry for peptide and protein sequence analysis

Proteins are involved in nearly every aspect of cellular function. In fact, the characterization of proteins has become such a significant part of modern biology, it has inspired a new discipline: proteomics—the classification of the protein complement expressed by the genome of an organism. Technology development has driven, and continues to drive, rapid evolution in this field. Seventeen years ago innovations in biomolecule ionization [electrospray (1) and matrix-assisted laser desorption/ionization (2), ESI and MALDI, respectively] placed mass spectrometry (MS) at the forefront of this emerging discipline. Although biological MS was pursued prior to this work, these techniques are generally credited for expanding the field substantially. With these tools, mass spectrometrists could for the first time, easily and robustly, convert condensed phase peptides or even whole proteins to intact gas phase ions.

Analysis of the acidic proteome with negative electron-transfer dissociation mass spectrometry.

We describe the first implementation of negative electron-transfer dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer and its application to high-throughput sequencing of peptide anions. NETD, coupled with high pH separations, negative electrospray ionization (ESI), and an NETD compatible version of OMSSA, is part of a complete workflow that includes the formation, interrogation, and sequencing of peptide anions. Together these interlocking pieces facilitated the identification of more than 2000 unique peptides from Saccharomyces cerevisiae representing the most comprehensive analysis of peptide anions by tandem mass spectrometry to date. The same S. cerevisiae samples were interrogated using traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic LC separations, positive ESI, and collision activated dissociation), and the resulting peptide identifications of the different workflows were compared. Due to a decreased flux of peptide anions and a tendency to produce lowly charged precursors, the NETD-based LC-MS/MS workflow was not as sensitive as the positive mode methods. However, the use of NETD readily permits access to underrepresented acidic portions of the proteome by identifying peptides that tend to have lower pI values. As such, NETD improves sequence coverage, filling out the acidic portions of proteins that are often overlooked by the other methods.

Front-End Electron Transfer Dissociation: A New Ionization Source

Electron transfer dissociation (ETD), a technique that provides efficient fragmentation while depositing little energy into vibrational modes, has been widely integrated into proteomics workflows. Current implementations of this technique, as well as other ion-ion reactions like proton transfer, involve sophisticated hardware, lack robustness, and place severe design limitations on the instruments to which they are attached. Described herein is a novel, electrical discharge-based reagent ion source that is located in the first differentially pumped region of the mass spectrometer. The reagent source was found to produce intense reagent ion signals over extended periods of time while having no measurable impact on precursor ion signal. Further, the source is simple to construct and enables implementation of ETD on any instrument without modification to footprint. Finally, in the context of hybrid mass spectrometers, relocation of the reagent ion source to the front of the mass spectrometer enables new approaches to gas phase interrogation of intact proteins.

Applications of linked scan procedures in investigating polyatomic ion/surface interactions

Abstract Tandem mass spectrometry (MS/MS) is applied to the study of polyatomic ion/surface interactions. The incident and emergent beams are tracked in two distinct types of experiments: (i) those in which the mass-to-charge ratio for one of the beams is fixed, and (ii) those in which both analyzers are scanned simultaneously. The former experiment yields either parent or daughter MS/MS spectra, based on an ion/surface interaction. The latter experiment can be used to obtain a mass spectrum which records either (a) all reflected ions or (b) all ions which undergo a specified change in either mass or charge. Examples will be given of parent spectra, of neutral loss spectra, and of reflection spectra. The reflection experiment does not have a strict analog among the more familiar gas-phase MS/MS experiments.

Determination of Positions, Velocities, and Kinetic Energies of Resonantly Excited Ions in the Quadrupole Ion Trap Mass Spectrometer by Laser Photodissociation

The effects on ion motion caused by the application of a resonance AC dipole voltage to the end-cap electrodes of the quadrupole ion trap are described. An excimer laser is used to photodissociate benzoyl ions, and its triggering is phase locked to the AC voltage to follow the motion of the ion cloud as a function of the phase angle of the AC signal. Resonantly excited ions maintain a coherent motion in the presence of He buffer gas, which dissipates energy from the ions via collisions. Maximum ion displacements, which depend upon the potential well depth (qz value), occur twice each AC cycle. Axial components of ion velocities are determined by differentiating the displacements of the distributions with respect to time. The experimental data show that these velocities are maximized when the ion cloud passes through zero axial displacement, and they compare favorably with results calculated using a simple harmonic oscillator model. Axial components of ion kinetic energies are low (<5 eV) under the chosen experimental conditions. At low values of q2 (≈ 0.2), the width of the ion distribution increases as the ion cloud approaches the center of the trap and decreases as it approaches the end-cap electrodes. This effect is created by compaction of the ion trajectories when ion velocities are decreased,