Christie Hunter

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How can I tell if my Spectral Peak is saturated on the TripleTOF system?

The key performance attributes for qualitative analysis are speed, mass accuracy, and resolution in both MS and MS/MS modes. For quantitative analysis, inter-scan dynamic range is important in order to maintain accuracy and linear response over a wide range of sample concentrations. The detection system on a TripleTOF system is a key component to achieving all these performance attributes on a single platform.  Dynamic Ion Transmission Control (ITC) is one feature on the TripleTOF systems where the MS ion current through the instrument is modulated scan by scan to ensure the ions reaching the detector are within the linear dynamic range of the detection system.  Here we will discuss how ion detection works and how to determine whether your signal is within the dynamic range of the detection system.

For TripleTOF and X500 QTOF systems

On these platforms signal from the detector is processed by a Time-to-Digital-Converter (TDC). Briefly, an ion impact at the detector produces an analog electrical pulse which is amplified and subsequently converted to a digital pulse. This is in turn recorded by the TDC and assigned a time bin to mark its time-of-flight. Statistics generated by summing results from many individual TOF extractions (typically occurring at 15 – 20 kHz) allow the use of a Poisson-based statistical correction (Saturation Correction).  This correction accounts for incidents of near-simultaneous ion impact events which would otherwise be counted as only a single ion. Saturation Correction reliably expands the linear dynamic range of TDC-based systems by more than an order of magnitude.

To determine whether your spectral peak of interest is within the linear range of the detector:

Open your file in PeakView software and open a single spectrum at the LC peak apex. Select the peak of interest and open the Graph Selection Window. The Sum Intensity number is the value you want to look at.

The pulser frequency of the method is important in the calculation of approximate Sum Intensity where saturation may be observed:

  • On TripleTOF 5600+ system, Max Sum Intensity for an MS/MS peak = Pulser frequency x 10

To determine the pulser frequency in Analyst for the mass range you are using, open Manual Tune, set the max m/z in the TOF Scan section, then record the pulser frequency from the Advanced MS tab (note it is also stored in the file information).

So , as an example, for proteomics experiments on a TripleTOF 5600 system operating up to m/z 1500 (Pulser Frequency = 17.5 kHz), we can expect to be quantitatively linear up to approximately 1.75e5 cps (Sum Intensity).

This is much more complicated to determine in MS mode at the spectral level because the data we see is the ITC corrected data. Please refer to the Dynamic Ion Transmission Control discussion for an easy way to roughly check at the chromatogram level.

Please note that these are just guidelines.   The actual saturation point will depend on a number of other factors that are more difficult to quantify.

For TripleTOF 6600+ systems

On these platforms, the detection system is ADC-based and has greater dynamic range. To measure ions on this type of detector system the amplified electrical pulse from the detector is sampled by the ADC and its area recorded. This measurement is compared to a known, pre-calibrated area for a single ion detection event so the number of ions that correspond to the incoming signal can be calculated. This allows the system to measure directly the number of ions impacting the detector without using a statistical correction. With the number of ions determined, this result is assigned a time bin to mark its time of flight.

Again, the pulser frequency of the method is important in the calculation of max sum intensity (see above for determining pulser frequency and Sum Intensity):

  • On TripleTOF 6600 system, Max Sum Intensity = Pulser frequency x 75

So , as an example, for proteomics experiments on a TripleTOF 6600 system operating up to m/z 1500 (Pulser Frequency = 17.5 kHz), we can expect to be quantitatively linear up to approximately 1.3e6 cps (Sum Intensity).

RUO-MKT-18-6120-A

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