Improving Quantitative Accuracy and Precision of Isobaric Labeling Strategies for Quantitative Proteomics Using Multistage (MS~3) Mass Spectrometry

摘要

Over the past decade, the field of proteomics has progressed from the identification of proteins in bio logical samples to protein quantification.1,2 Several strategies have been developed, one of which is sta- ble isotope labeling, where samples are labeled and the relative abundances of proteins are calculated across two or more sam- ples. There are two major types of isotope labeling techniques: 1) metabolic labeling-SILAC (stable isotope labeling with amino acids in cell culture), and 2) chemical labeling-tandem mass tags (TMT~?, Thermo Fisher Scientific [Waltham, MA]) and isobaric tags for relative and absolute quantitation (iTRAQ~?, AB SCIEX [Framingham, MA]). These two techniques differ in where the labeling occurs. In metabolic labeling, the labeling takes place at the metabolic level, whereas chemical labeling is done after the samples have been enzymatically digested, typically with trypsin. Chemical labeling has become more popular since it is easier to perform and up to eight samples can be multiplexed. Each sample is labeled with a different label and the samples are mixed and subjected to liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis.