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Novel methods for the generation of peptide, phosphopeptide and protein standards are described. Inductively coupled plasma mass spectrometry (ICP-MS) is used for quantitative detection, and electrospray ionization mass spectrometry (ESI-MS) is used for molecular characterization and purity control. Stable-isotope-labeled phosphopeptide standards are prepared by chemical synthesis with the introduction of phosphorus as an ICP-tag, and are accurately quantified using a combined y-split µLC-[ICP/ESI]-MS system. In addition, phosphopeptide standards can be used as the starting materials for the production of corresponding peptide standards via quantitative enzymatic dephosphorylation. This conversion step is controlled for completeness by ESI-MS. Stable-isotope-labeled protein standards are produced by cell-free synthesis with the stoichiometric introduction of selenium in the form of L-SeMet as an ICP-tag. The trueness of absolute standard quantification via ICP-MS and Se detection is not biased by the presence of other proteins since L-SeMet is exclusively present in the standard protein, where it replaces L-Met. RISQ (recombinant isotope-labeled and selenium quantified) protein standards contain both SeMet and stable-isotope-labeled amino acids of choice. Protein standards with SeMet but without stable isotope labels (recombinant selenium quantified, RSQ) and protein standards with only stable isotope labels (recombinant isotope-labeled and quantified, RIQ) are also described. In summary, the production of novel proteomics standards is proposed characterized by precision ≤5% and controllable trueness of their quantification.

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