Large-Scale Proteome and Phosphoproteome Quantification by Using Dimethylation Isotope Labeling
Protein characterization alone is usually not enough to elucidate most biological processes. Although thousands of proteins can be identified in one proteomic experiment, it is difficult to relate these proteins with their biological functions. The expres
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Large-Scale Proteome and Phosphoproteome Quantification by Using Dimethylation Isotope Labeling
4.1 Introduction Protein characterization alone is usually not enough to elucidate most biological processes. Although thousands of proteins can be identified in one proteomic experiment, it is difficult to relate these proteins with their biological functions. The expression levels of proteins within a living organism are reflections of the different physiological and pathological states. Conventional protein quantification technologies such as western blot (WB) are low throughput and can only quantify one protein in each experiment. Therefore, comprehensive proteome quantification in certain depth is an important direction in the development of proteomic technologies and is considered as the bridge for the gap between proteins and their biological function [1–7]. On the other hand, there are more than 300 types of posttranslational modifications (PTMs) that can dynamically modify the whole proteome, which makes the protein sample even more complex. The occupancy level of a PTM on the specific site of a protein is also critical to the biological function of the protein in the regulation of different physiological and pathological processes, such as the protein phosphorylation is related to the signal transduction in many pathways activation and protein glycosylation is related to cell-to-cell recognition [8–10]. Therefore, comprehensive quantification of proteome PTMs is also another important task for current proteomic analysis. Two strategies are feasibly developed for the comprehensive proteome quantification in mass spectrometry (MS)-based shotgun proteomics. The first is the labelfree approach, which obtains the relative quantity of each peptide among different samples by comparing the corresponding peak intensity in parallel nanoflow liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses [11, 12]. The advantage of the label-free approach is that no isotope labeling is required, and multiple protein samples can be compared simultaneously. However, the quantification accuracy largely depends on the reproducibility of nanoflow LC–MS/MS analysis. The other is the stable isotope labeling strategy, which introduces different mass differentiate isotope tags into different samples at first, and
F. Wang, Applications of Monolithic Column and Isotope Dimethylation Labeling in Shotgun Proteome Analysis, Springer Theses, DOI: 10.1007/978-3-642-42008-5_4, © Springer-Verlag Berlin Heidelberg 2014
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4 Large-Scale Proteome and Phosphoproteome Quantification
then the samples are mixed together and analyzed by LC-MS/MS simultaneously. The relative quantity of each peptide among different samples is obtained by comparing the intensity of specific isotopic peaks in the corresponding MS spectrum [13–15]. Stable isotope labeling with amino acids in cell culture (SILAC) introduces isotope tags by in vivo cell culture, which can compare the expression levels of thousands of proteins in a single label
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