ITRAQ-Labeled Yeast Peptide Clean-Up Using a Reversed-Phase Column

Abstract

INTRODUCTIONA commercial reagent, known as the isobaric tag for relative and absolute quantification (iTRAQ), makes it possible to analyze multiple samples simultaneously. The ability of iTRAQ to compare relative protein abundances across as many as eight samples is a significant advantage over other stable isotope strategies, such as stable isotope labeling by amino acids in cell culture (SILAC) and isotope-coded affinity tag (ICAT). The iTRAQ protocol set compares the proteome of Saccharomyces cerevisiae under two different metabolic states (fermentation versus respiration) with the goal of determining the proteins involved in each of the activated pathways by measuring their relative abundances using iTRAQ. Before using isoelectric focusing (IEF) to fractionate the iTRAQ-labeled peptides, the sample needs to be cleaned up because it contains a high concentration of buffer (500 mM triethylammonium bicarbonate), excess reagents from the reduction and alkylation of the proteins, and degradation products of the iTRAQ reagents. All of these are incompatible with IEF. To remove them, the sample is passed over a reversed-phase C18 column. This protocol describes this procedure.