Relative Quantification of Proteome via Partial Metabolic Heavy Water Labeling

Abstract

Novel Aspect A new method for relative quantification of proteome via partial metabolic heavy water labeling

Introduction Various methods via the application of stable isotope labeling have been developed for relative protein quantification based on mass spectrometry (MS). Compared with chemical labeling methods, metabolic labeling approaches have the advantage of reducing technical variability thanks to the option of mixing light and heavy variants prior to all sample preparation steps. SILAC, the most representative metabolic labeling method, has successfully been applied in a number of quantitative proteomics for cell culture and small organisms. The application of metabolic labeling in higher organisms, however, is challenging because tightly controlled diet conditions are essential for satisfactory labeling performance. To overcome this obstacle, a widely applicable relative protein quantification method via partial metabolic labeling of heavy water (2H2O) is proposed.

Methods Proteins from HeLa and SW480 cells cultured in normal and 5% 2H2O enriched media for 48 hours were mixed in various mixing ratios based on their protein concentrations, and separated using GELFrEE. After tryptic digestion, peptides were analyzed with LC-MS/MS followed by measurement of their isotopic distributions. Using the isotopic distributions of unlabeled and labeled peptides, a mixed isotopic distribution library is generated by calculating the mixing ratios from 99:1 to 1:99. The relative abundances of unlabeled and labeled peptides in a mixed sample are calculated from subtle changes in the isotopic distributions in a 1:1 mixture. Our method was evaluated with respect to accuracy and precision by comparing it with the SILAC method.

Preliminary data Quantification of HeLa cell proteins in various mixing ratios was carried out to evaluate the accuracy and precision of the relative quantification based on the 2H2O labeling. Quantification of 1:1 mixed HeLa cell proteins in three biological and technical replicates showed high reproducible results between them. Peptides with quantitative results close to the mixing ratio revealed two key characteristics for the quality of the relative quantification, which are the number of detectable mass isotopomers and the degree of deuterium incorporation. These two parameters provided the criteria that successfully filter poorly quantifiable peptides and helped to improve the quality of quantification in terms of accuracy and precision. To investigate the dynamic range of our quantification method, HeLa cell extracts were mixed in various mixing ratios from 10:1 to 1:10. Most of the quantitative results for HeLa cell extracts mixed from 10:1 to 1:10 showed the expected relative abundances. If the mixing ratio was too large or too small, however, quantification was not successful because of indistinguishable isotope distribution difference between similar mixing ratios. In a comparative experiment with SILAC using SW480 cells being mixed in a 1:1 ratio, the numbers of peptides which have fold change values within 2-fold and 0.5-fold intervals were comparable to each other. Similar to peptide quantification, both methods showed similar protein quantification performance.