Workflow Overview for Targeted Quantitation

While targeted peptide quantitation has historically been the domain of triple-quadrupole mass spectrometers, recent advances in Thermo Scientific Orbitrap-based instruments have made them equally well-suited to quantitative experiments. On these instruments, MS1 level scans are used to monitor for the presence of the targeted species. When the mass of a known (or predicted) peptide is identified in an MS1 spectrum, the instrument triggers an MS/MS scan for confirmation of the target’s identity. The MS1 abundance of the targeted ion is used for absolute or relative quantitation based on its accurate mass. Alternatively, targeted quantitation can be done using dedicated MS/MS (or MSⁿ) scans. In this case, target quantitation is based on the abundance of one, or several, MS/MS (or MSⁿ) fragments. The dramatic increase in resolution and mass accuracy afforded by Orbitrap^TM detection facilitates sensitive and selective quantitation at both the MS and MSⁿ levels. Targeted peptide quantitation can be applied to both labeled and unlabeled targets.

Mass Spectrometry Workflow for Targeted Quantitation

Recently, high-resolution, accurate-mass detection has shown tremendous potential for targeted protein/peptide quantitation. The unmatched resolution afforded by Orbitrap technology allows for discrimination between the target ion species and very closely spaced interferences in complex sample matrices. This high selectivity greatly contributes to lowering the limits of detection and to increasing the accuracy and precision of quantitation.

The first step to developing a targeted quantitation method is to determine which peptide species are most representative of the respective targeted proteins. Care should also be given to select peptides that possess favorable properties for MS analysis. These peptides can be selected from discovery data, or from a priori knowledge of the peptides expected to be present. Thermo Scientific Pinpoint software automates this process using previously acquired data or known target protein sequence information.
For targeted analysis in complex (i.e. whole proteome) samples, it is insufficient to rely on detection and quantitation of a single ion in the MS1 domain alone. This is because, despite the HR/AM features afforded by Orbitrap analysis, the presence of numerous isobaric peptide species can render essentially all MS1 signals ambiguous. Consequently, when a candidate signal is detected in the MS1 data, it’s identity must be confirmed by MS/MS analysis. Targeted peptides are identified and quantified using selected ion monitoring (SIM) and then confirmed with data-dependent or data-independent scheduled MS/MS. The most important advantage of SIM is improvement in LODs. This derives from the fact that in SIM, the entire Orbitrap ion capacity is devoted to a narrow range of ion masses around the target; consequently a signal that might be too faint to be well characterized from a full MS1 scan may be well characterized by a SIM scan. Typically, the complexity of biological samples requires confirmation of the target peptide’s identity using MS/MS, even when HR/AM precursor scan information is present. This is done using either a CID or HCD scan on hybrid ion trap-Orbitrap instruments or an HCD scan on hybrid quadrupole-Orbitrap instruments. The advantage of using a CID scan with ion trap detection in the hybrid instrument is the exceptional sensitivity provided by ion trap detection, which can provide high-quality spectra even for ultra-low-level targets.

Hybrid Orbitrap and Q Exactive instruments are routinely used in discovery-based proteomics experiments for both identification and relative quantitation. Using the same instruments for targeted quantitation experiments greatly streamlines the transition from discovery workflow to targeted workflow. The experimental advantages realized in discovery proteomics carry over to targeted quantitation. These include:

• High mass accuracy: Allows for greater selectivity, accuracy and precision of quantitation at the same time increasing confidence in the identity of the quantified target.

• Ultra-high resolution: Increases selectivity and lowers detection limits for targeted analyte

• High intra-scan dynamic range: Increases linearity and dynamic range of the quantitation experiment

• Low detection limits as ions of a target peptide may be accumulated/enriched in the mass spectrometer prior to detection.
   

• High sensitivity MS/MS: Both CID and HCD can be used for identity confirmation, with CID in the ion trap being the most sensitive MS/MS mode, allowing target confirmation right at the detection limit of the SIM scan.