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Accurate quantification of proteins is important for a wide range of questions in molecular and cell biology, systems biology, or clinical research. Depending on the specific question asked, either relative quantitative changes across multiple samples (relative quantification) or absolute protein concentrations of proteins in a particular sample (absolute quantification) are required. Absolute quantification is beneficial, for example, in studies on protein complex stoichiometries, mathematical modeling of biological processes, clinical biomarker development, or for comprehensive inter-experimental, inter-laboratory and inter-organism comparisons. In recent years, targeted mass spectrometry via selected reaction monitoring (SRM) has proven suitable for reproducible, precise and sensitive absolute quantification of predetermined sets of proteins. In this chapter we provide a general overview of the most commonly applied absolute quantification strategies with SRM. These include the use of stable-isotope-labeled peptide and protein standards, as well as label-free strategies. Advantages and limitations of each workflow are presented and compared. Finally, important challenges and pitfalls specific for absolute protein quantification are highlighted and future perspectives for the field of targeted proteomics are discussed.

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