QqOrbitrap (QExactive, Thermo)


The Q Exactive is a hybrid mass spectrometer combining the performances of a quaripole for the precise selection of precursors and an orbitrap for a high resolution and an accurate mass (HR/AM) detection.

This configuration allows to increase the scanning speed and offers new multiplexing possibilities, making it compatible with ultra fast chromatography.

The Q Exactive is adapted to routine bottom-up analyses for the identification of proteins as well as semi-quantitative analyses (isotopic labeling) and targeted analyses such as PRM (parallel reaction monitoring) thanks to the high resolution of the orbitrap.

Application domains

  • Identification of proteins from a simple mixture (direct injection) or from a complex mixture after a 1D or 2D LC separation.
  • Searching for exact masses of modifications
  • Quantification of peptides following an ITRAQ,ICAT, TMT or SILAC labeling

Measurement principle

The configuration QqOrbitrap combines two advantages : the first quadripole allows to rapidly select ions of interest since the application of the voltage is very brief and since there’s no scanning, and the second quadripole set to HCD allows to fragment the peptides on a similar time scale. Moreover, the in-space separation and the possibility of working at high resolution in both MS and MS/MS modes opens the field to multiplex analyses.

The ions formed in the electrospray source (the only source available commercially for this instrument) are accelerated and enter the orbitrap analyzer to be separated according to the mass/charge ratio. The orbitrap is composed of a hollow electrode inside of which a spindle-like electrode is co-axially placed. The specific forms of these two electrodes allows the application of a quadripolar electrostatic field following the z axis of the electrodes. The ions are injected tangentially to the central electrode and trapped around it by the electrostatic force that compensates the centrifuge forces. The movement of the ions can thus be decomposed as such: a circular movement around the central electrode in the (xy) plane and an oscillatory back and forth movement following the z axis. In particular, ions with a given m/z are on the same circular trajectory that oscillates with a frequency f that is independent of the speed and the energy of the ions. Just like with the FT-ICR, the current induced by these oscillations allows to access the m/z values via a Fourier transform. A modification of the signal treatment has been implemented. The transients detected by the orbitrap are treated by optimized Fourier transform (eFT™) to convert the temporal signal into a frequency then into an m/z ratio. These calculations have been detailed by [ ]. The calculation of the Fourier transform uses complex numbers that can be assimilated to the signal’s amplitude and phase, or by a real part and an imaginary part in mathematics. Since the initial phase of the ion packet depends of the initial parameters at the moment of the injection in a relatively complex way, the FT spectra are generally interpreted in the so-called "amplitude" mode, this is to say the phase information is not taken into account. However, with the orbitrap, the excitation mechanism by injection provides an initial phase that is independent of the m/z ratio in a first approximation. This synchronization allows to convert the spectra in a way that the real part of the data can be exploited, which permits to obtain finer peaks. Practically, this eFT mode allows to enhance the mass precision and peak profiles by using the real and imaginary parts. The synchronization is better if the detection starts right after the end of the injection. This principle has been applied by decreasing the delay between the injection and the detection from around 10 ms to a fraction of a ms, which is equivalent to increasing the resolution by factor slightly less than 2 for the same transient recording (except for proteins whose signal degrades rapidly by a factor of 1.4 by collision with residual gas molecules). The time is takes to treat the data remains inferior to the acquisition and injection time. However, to be efficient, the eFT demands a very good electronic synchronization and remains sensitive to information transfer delay. The measurement precision remains comparable to that obtained by classic FT.

Multiplexing

The precursor selection that’s done "in-space" (in the quadripole) is extremely rapid. This allows a complex association of MS and MS/MS operations followed by a high resolution analysis in the orbitrap (see [193]).

In Selected Ion Monitoring (SIM) a narrow mass range is accumulated to increase the signal to noise ratio of an ion of interest. SIM analyses are possible in the LTQ-Orbitrap configurations, but are not often used. Indeed (1) the isolation in SIM mode in the linear trap is relatively slow, (2) the number of ions that can be isolated is limited by space charge effects, (3) the analysis in the orbitrap of a single SIM scan is long. The Q Exactive doesn’t suffer from the same limitations. In SIM mode the C-trap is used as an ion storage zone and is filled with the desired number of ions. In multiplex analyses in SIM mode, up to 10 ions of different m/z ratios can be stored in the C-trap before being simultaneously injected and analyzed in the orbitrap (see figure 1). Since fill times are shorter than transient detection times, the orbitrap is used in a much more efficient way in multiplex analyses.

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See also...

MALDI-TOF/TOF (5800, AB SCIEX)

Application domains Identification of proteins in simple mixtures (purified by 1D or 2D acrylamide gels) or in complex mixtures after an LC (...) 

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LTQ-FT-ICR (Thermo)

Domain of applications Identification of proteins in simple mixtures (direct injection), on in complex mixtures following a 1D or 2D LC (...) 

> More...