Global Proteomic Profiling of Embryonic Stem Cells Using iTRAQ Isobaric Tags with LC-MS/MS Quantification.

In this methods chapter, we describe the use of isobaric tags for relative and absolute quantification (iTRAQ) for the differential expression analysis of global proteins between embryonic stem cell samples. This protocol describes how proteins are collected from cell culture, digested and prepared so that peptides are labeled with these isobaric tags. Labeled digests are pooled, fractionated offline, and quantified using liquid chromatography-mass spectrometry (LC-MS). This offline fractionation allows for a greater separation and thus increased identification/quantification of peptides. This combined method enables large-scale, deep penetration into the proteome of embryonic stem cells. During quantification, the relative intensities of label-derived reporter ions represent the relative amount of peptide in each sample. Using search algorithms that integrate the generated data for the identified and quantified peptides allows the relative quantification of proteins in the samples. The isobaric tags can be used in a 4 or 8 multiplexed manner; however, using an 8-plex experimental setup allows for the simultaneous analysis of biological and technical replicates within the same mass spectrometry run, thus minimizing experimental variation and increasing the confidence in any identified expression differences.