Absolute Quantitation of Protein Contents Based on Exponentially Modified Protein Abundance Index by Mass Spectrometry

摘要

The present inventor has established protein abundance index (PAI, π) to determine the protein contents in a protein mixture solution using nanoLC-MSMS data. Digested peptides were analyzed by nanoLC-MS/MS and the obtained results were applied to a Mascot protein identification algorism based on tandem mass spectra. PAI is defined as the number of observed peptides divided by the number of observable peptides per protein. PAI from different concentrations of serum albumin showed linear relationship to the logarithm of the protein concentration. This was also valid for 47 proteins in a mouse whole cell lysate analyzed by single run of nanoLC-MS/MS. On the other hand, Mascot protein scores as well as the number of identified peptides per protein were less correlated to the protein abundance. For absolute quantitation, PAI was converted to exponentially modified PAI (EMPAI, mπ), which is proportional to protein contents in the protein mixture. For the 47 proteins in the whole lysate, the deviation percentages of the EMPAI-based concentrations to the actual values were within 63% in average. EMPAI was successfully applied to comprehensive protein expression analysis and performed a comparison study between gene and protein expression in an HCT116 human cancer cells. Accordingly, the present invention provides a method and a computer program for quantifying the protein contents based on the protein abundance index.