Orbitrap Illustration

Targeted Quantitation

The objective of targeted protein quantitation experiments is to determine the protein and/or peptide expression levels of known targets in biological systems. The experiment may be designed to determine either the relative levels of the target species or the absolute levels. The scope of these experiments can range from the analysis of individual samples in a research environment to the assessment of thousands of samples in a clinical research setting.  Mass spectrometry-based targeted quantitation requires a priori knowledge of the molecular targets, as well as of the general properties of the samples in which they are contained.  At a minimum, the scientist must know the molecular weight of the targeted species. Knowledge of additional properties of the targets, such as their LC elution times, their expected range of expression levels, and their dynamic range, as well as knowledge of the characteristics of the background matrix, will all help greatly in designing a successful targeted quantitation experiment.  Demand for targeted quantitation experiments among scientists and clinicianshas undergone explosive growth in recent years. 

Fortunately, advances in mass spectrometry hardware, including high-resolution, accurate-mass (HR/AM) instruments, sample preparation methodologies, and data processing software have now made it possible to develop robust targeted quantitation workflows suitable for analysis of hundreds to thousands of targets in a single analysis.

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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 OrbitrapTM 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.