Tailored use of targeted proteomics in plant-specific applications

Abstract

Targeted proteomics comes in different flavors and the literature is full of associated technical terms such as selected reaction monitoring (SRM), multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), and accurate inclusion mass screening (AIMS) (reviewed in e.g. Picotti and Aebersold, 2012; Boersema et al., 2015; Gillet et al., 2016; Borràs and Sabidó, 2017). Recently, the data-independent acquisition method sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was introduced as a targeted proteomic method even though it differs considerably from the others (see below) (Gillet et al., 2012; Röst et al., 2014). The underlying rationale of all targeted proteomics approaches is the specific identification, characterization and quantification of a limited set of pre-defined peptides or proteins in an MS analysis. Nature Methods elected targeted proteomics method of the year in 2012 because it has great advantages over untargeted (referred to as “survey” or “discovery”) proteomics with respect to identification sensitivity and quantification reproducibility. In discovery proteomics, scans are performed over the full accessible mass range to select precursor ions for fragmentation (MS/MS). This increases the time for the identification, selection and fragmentation of precursors (duty cycle) and the dynamic range of different precursor abundances that need to be handled. Because proteomes are highly complex, precursor selection is a stochastic process making MS/MS-based peptide identifications inherently difficult to reproduce (Figure 1). In targeted approaches, the MS only scans for a set of pre-defined masses thus eliminating the stochastic nature of precursor selection and reducing duty cycle times and dynamic range constraints. Additionally, signal-to-noise ratios improve significantly, and acquisition times for peptides within a limited mass range (dwell times) can be increased to increase sensitivity (Figure 1; Borràs and Sabidó, 2017; Hart-Smith et al., 2017). Taken together targeted MS allows testing specific hypotheses with high accuracy at high reproducibility, which has led Picotti and colleagues to designate targeted proteomics as the bridge from large-scale data acquisition to the “scientific method” (Picotti et al., 2013a).