Improving the precision of quantitative bottom-up proteomics based on stable isotope-labeled proteins

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.