In addition to data-dependent approaches, targeted methods also exist whereby specific ion transitions can be monitored. These various targeted approaches are summarized in Figure 1.1, each of which has been employed to characterize post-translationally modified peptides.
Precursor ion scanning (PIS) is a sensitive mode of mass spectrometric operation primarily performed on triple quadrupole instruments, which has been
Figure 1.1 The selected reaction monitoring technique. Molecular ions of a specific analyte are selected in Q1 and fragmented in Q2. electrospray ionization (ESI). Molecular ions of one or several contaminants are isolated and fragmented together. A specific fragment ion from the target analyte (transition) is selected in Q3 and guided to the detector. The number of target fragment ions is counted over time, resulting in an SRM trace for each transition. On the far right, cycles through three transitions, corresponding to three different fragments of the target analyte, and the corresponding three SRM traces are shown. Source: Picotti and Aebersold 2012 . Reproduced with permission from Macmillan Publishers Ltd.
employed for the analysis of predefined PTMs. In PIS, the third quadrupole of a triple quadruple mass spectrometer is fixed on a selected m/z, typically that being a neutral loss ion, for example, 79 Da for a phosphate anion observed in negative ion mode of detection, whereby the (PO3) species is derived from the CID of phosphorylated moieties . This method is highly selective and sensitive and has been applied to other PTMs beyond the analysis of phosphopep- tides. Another targeted method traditionally employed for identification of post-translationally modified peptides is neutral loss scanning (NLS) . NLS experiments monitor all pairs of precursor ions and product ions that differ by a constant neutral loss consistent with the PTM of interest. However, with the exponential improvements in speed and sensitivity for instruments such as the orbitrap and Q-TOF, these methods are less commonly employed than several years ago.