As discussed earlier in Section 1.6, tandem mass spectrometry experiments are useful for acquiring fragment ion spectra of the ion of interest which, for biomolecules, is typically used for peptide sequencing, discussed later in Section 2.4.2. When continually infusing a pure analyte into the mass spectrometer, the intact and fragment ion spectra can be easily acquired separately by activating and deactivating fragmentation in the collision cell, to obtain both the intact mass measurement, and the fragment ion spectra necessary to analyse the sample. However, when LC is used to separate complex mixtures prior to MS analysis, the ion is only introduced into the mass spectrometer for the brief period that it elutes off the LC column, making rapid acquisition of both the intact, and the fragment ion spectra for each molecule challenging. To tackle this issue, instrument methods have been developed to allow rapid automated switching between high and low energies in the collision cell, allowing acquisition of both MS, and tandem MS data of ions as they are introduced into the mass spectrometer from online LC separation. Generally, there are two main acquisition modes that achieve this: data dependent, and data independent, acquisition.
1.8.1 Data dependent acquisition (DDA)
In data dependent acquisition modes (DDA), the spectra for intact molecular ions are first acquired by deactivating the fragmentation, and allowing a full m/z scan of all ions eluting from the LC column at that time. A user-defined number of the most intense m/z ions from the initial scan, sometimes called the MS1 or precursor ion scan, are then sequentially selected by the first mass analyser for fragmentation and spectral analysis by the second mass analyser. When complete, another full m/z scan is acquired at the new, now longer, LC retention time, and the cycle is repeated for as long as the LC elution takes place (Figure 1.22). Typically, a dynamic exclusion time
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is applied such that the same m/z value is not repeatedly selected if it remains at high intensity after the DDA cycle finishes. This is to prevent continual fragmentation of the same ions, and to more effectively utilise the available MS/MS time to fragment ions of different m/z.
However, DDA modes for LC-MS/MS present a trade-off between the speed, and depth of the analysis. Whilst sequentially selecting and fragmenting ions, full scan intact MS data cannot usually be acquired, thus some ions eluting from the LC column may be missed if the DDA cycle takes too long. Conversely, making the cycle shorter by reducing the number of different m/z ions to select and fragment, allows the DDA cycle to repeat more frequently, but at the cost of narrowing the analysis to only a few of the most intense ions. That said, the use of product ion scans and m/z selection in DDA make it a very popular MS/MS method, as only ions fragmented from the precursor are present in the tandem MS spectra, drastically simplifying data analysis.
Figure 1.22 Operating procedure of data dependent acquisition (DDA) in LC-MS experiments. A full MS scan is first performed to determine the most abundant ions in the spectra at the current LC retention time. The top x most intense ions, x being a parameter tuneable by the operator, are then sequentially selected by the first mass analyser (usually a quadrupole) where product ion scans are used to obtain tandem MS spectra for each ion selected. When x is reached, a full scan is performed again. This process is repeated across the entire LC gradient elution.
35 1.8.2 Data independent acquisition (DIA)
Data independent acquisitions (DIA) involve rapid switching between high and low energy modes without any m/z selection (Figure 1.23). The result is that co-eluting precursor ions are fragmented together, generating complex tandem MS spectra with fragment ions present from multiple precursors. Whilst this method allows both deep and rapid analysis of the sample, acquiring MS1 and MS2 spectra quickly for ions as they elute from the LC column, the data analysis is far more complicated, as assigning which fragment ions in the high energy spectra belong to which precursor ions in the MS1 spectra can be challenging.
Figure 1.23 Operating procedure for data independent acquisition (DIA) in LC-MS experiments. The instrument switches rapidly between high and low energy modes without any precursor m/z selection. Co-eluting precursors are fragmented together generating highly complex MS/MS data.
One common method of DIA data analysis, termed MSE, matches precursor ions to
their respective fragment ions by retention time alignment [56]. In this method, shown
in Figure 1.24, the change in intensity of the precursor and fragment ions over the course of the LC separation is used to determine their respective retention times. Fragment ions will have the same retention time as the precursor ion from which they are produced, so these data can be used to reconstruct MS/MS data and assign fragment ions to their corresponding intact molecular ions.
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Figure 1.24 Data analysis in DIA-MSE. The retention times of precursor ions are matched to the
retention times of the fragment ions. These data are then used to reconstruct the tandem MS spectra which correspond to particular precursor ions.