Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates.

Proteome coverage and peptide identification rates have historically advanced in line with improvements to the detection limits and acquisition rate of the mass spectrometer. For a linear ion trap/Orbitrap hybrid, the acquisition rate has been limited primarily by the duration of the ion accumulation and analysis steps. It is shown here that the spectral acquisition rate can be significantly improved through extensive parallelization of the acquisition process using a novel mass spectrometer incorporating quadrupole, Orbitrap, and linear trap analyzers. Further, these improvements to the acquisition rate continue to enhance proteome coverage and general experimental throughput.

The Orbitrap: a new mass spectrometer.

Research areas such as proteomics and metabolomics are driving the demand for mass spectrometers that have high performance but modest power requirements, size, and cost. This paper describes such an instrument, the Orbitrap, based on a new type of mass analyzer invented by Makarov. The Orbitrap operates by radially trapping ions about a central spindle electrode. An outer barrel-like electrode is coaxial with the inner spindlelike electrode and mass/charge values are measured from the frequency of harmonic ion oscillations, along the axis of the electric field, undergone by the orbitally trapped ions. This axial frequency is independent of the energy and spatial spread of the ions. Ion frequencies are measured non-destructively by acquisition of time-domain image current transients, with subsequent fast Fourier transforms (FFTs) being used to obtain the mass spectra. In addition to describing the Orbitrap mass analyzer, this paper also describes a complete Orbitrap-based mass spectrometer, equipped with an electrospray ionization source (ESI). Ions are transferred from the ESI source through three stages of differential pumping using RF guide quadrupoles. The third quadrupole, pressurized to less than 10(-3) Torr with collision gas, acts as an ion accumulator; ion/neutral collisions slow the ions and cause them to pool in an axial potential well at the end of the quadrupole. Ion bunches are injected from this pool into the Orbitrap analyzer for mass analysis. The ion injection process is described in a simplified way, including a description of electrodynamic squeezing, field compensation for the effects of the ion injection slit, and criteria for orbital stability. Features of the Orbitrap at its present stage of development include high mass resolution (up to 150,000), large space charge capacity, high mass accuracy (2-5 ppm), a mass/charge range of at least 6000, and dynamic range greater than 10(3). Applications based on electrospray ionization are described, including characterization of transition-metal complexes, oligosaccharides, peptides, and proteins. Use is also made of the high-resolution capabilities of the Orbitrap to confirm the presence of metaclusters of serine octamers in ESI mass spectra and to perform H/D exchange experiments on these ions in the storage quadrupole.

Interfacing the orbitrap mass analyzer to an electrospray ion source.

The orbitrap mass analyzer employs the trapping of pulsed ion beams in an electrostatic quadro-logarithmic field. This field is created between an axial central electrode and a coaxial outer electrode. Stable ion trajectories combine rotation around the central electrode with harmonic oscillations along it. The frequencies of axial oscillations and hence mass-to-charge ratios of ions are obtained using fast Fourier transform of the image current detected on the two split halves of the outer electrode. This work proves that such a trap could be coupled to a continuous, electrospray, ion source. Such a coupling necessitated the development of an rf-only quadrupole for external accumulation of ions and their injection in very short (< 1 micros) ion bunches. Along with good sensitivity, this mass spectrometer provides mass resolving power up to 150,000 fwhm, mass accuracies within a few parts per million, and relative mass range up to 8-fold. The maximum number of ions available for analysis is limited by the space-charge capacity of the accumulation quadrupole.