Tools and Analyses for Differential Label-Free Proteomics Using Mass Spectrometry

Abstract

The comparative measurement of protein abundance is a powerful method to detect changes in the biological dynamics of cells and tissues. Shotgun proteomics has proven to be a method where a wide range of proteins can be characterized in a single experiment by analysis of their peptide digests using micro-capillary liquid chromatography coupled to mass spectrometry (μLC-MS). We have the CRAWDAD software tool for label-free relative quantitation between samples using peptide signals in μLC-MS data associated with their identifications from MS/MS data. CRAWDAD discovers features for quantification in μLC-MS data, lowers chromatographic and signal variance across replicates, and finds statistically significant changes in peptide abundance between samples. We have applied this tool toward model systems of induced expression of the <italic>Lac</italic> operator in <italic>E. coli</italic> to qualitatively assess the protein changes detected. A controlled set of changes using an <italic>E. coli</italic> protein digest spiked in at changing levels over a constant background of human proteins assesses the precision and accuracy of the detected changes in peptide and protein levels, and showed our results to be superior to label-free quantitation using spectral counting.