title: Preparation of heteroelement-incorporated and stable isotope-labeled protein standards for quantitative proteomics.
abstract: A major obstacle for further development of quantitative proteomics is the lack of accurately quantified protein standards. The following protocol describes innovative methods for the production of stable isotope-labeled protein standards. Their production is achieved by cell-free protein synthesis, which enables simultaneous incorporation of selenomethionine and stable isotope-labeled amino acids. The selenium tag allows sensitive and accurate quantification by inductively coupled plasma mass spectrometry (ICP-MS). The stable isotope label allows internal standardization in mass spectrometry-based proteomics by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Both label types can be placed within a single protein RISQ standard (recombinant isotope-labeled and selenium quantified) or can be distributed over two types of related RSQ and RIQ standards for the same target protein (recombinant selenium quantified and recombinant isotope-labeled and quantified). The combination of cell-free synthesis as production method with ICP-MS and ESI-MS/MS as detection methods results in protein standards, which are quantified at an outstanding level of accuracy.
authors.affiliation: Molecular Structure Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg, 69120, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Konopka
authors.forename: Nico
authors.initials: N
authors.lastname: Zinn
authors.forename: Christina
authors.initials: C
authors.lastname: Wild
authors.forename: Wolf D
authors.initials: WD
authors.lastname: Lehmann
mesh_terms: D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D007553:Isotope Labeling; D011506:Proteins / Q000235:genetics / Q000378:metabolism* / Q000592:standards; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D011506:Proteins