Absolute Quantification of Yeast Proteins Using Synthetic Peptide Standards and TMT labeling

The absolute protein amounts for the selected yeast ribosomal proteins (RPs) were determined using the standard peptides and TMT labelling. The range of the standard tryptic peptides were selected and synthesized based on the preliminary data-dependent experiments. The synthetic peptides were mixed and four aliquots containing different amounts of the peptides were labelled each with a different TMT labelling reagent. The yeast cell lysate was digested and labelled in parallel. The labelled cellular and standard samples were combined, and the absolute peptide amounts in the lysate were calculated by comparison to the known amounts in the standard TMT channels.