title: Improving the precision of quantitative bottom-up proteomics based on stable isotope-labeled proteins.
abstract: Stable isotope dilution-based quantitative proteomics with intact labeled proteins as internal standards in combination with a bottom-up approach, i.e., with quantification on the peptide level, is an established method. To explore the technical precision of this approach, calmodulin-like protein 3 was prepared in non-labeled (light) and SILAC-type labeled (heavy) form by cell-free synthesis, mixed, digested with trypsin, and analyzed by UPLC-ESI-MS. In total, 16 light/heavy peptide pair ratios were determined. Pair-wise comparison of ratios of 12 peptides selected according to S/N ratios >50 revealed that the majority exhibited ratios, which were different at a high level of statistical significance (p < 0.001). HPLC-MALDI-MS ratio data confirmed this observation, thus excluding the ionization method as a source of the observed ratio differences. Variation of the digestion time from 0.25 to 4 h showed that the light/heavy ratios of most peptides decrease with time, indicating a kinetic isotope effect leading to preferred cleavage of light calmodulin-like protein 3. The subset of peptides with statistically identical ratios resulted in an average ratio with a RSD of 1.0 %. The light/heavy ratio calculated on the basis of these peptides probably provides the most accurate molar protein ratio.
authors.affiliation: Molecular Structure Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Konopka
authors.forename: Martin E
authors.initials: ME
authors.lastname: Boehm
authors.forename: Marion
authors.initials: M
authors.lastname: Rohmer
authors.forename: Dominic
authors.initials: D
authors.lastname: Baeumlisberger
authors.forename: Michael
authors.initials: M
authors.lastname: Karas
authors.forename: Wolf D
authors.initials: WD
authors.lastname: Lehmann
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: C082965:CALML3 protein, human; D002147:Calmodulin