Isobaric Labeling-Based Relative Quantification in Shotgun Proteomics

Abstract

Mass spectrometry plays a key role in relative quantitative comparisons of proteins in order to understand their functional role in biological systems upon perturbation. In this review, we review studies that examine different aspects of isobaric labeling-based relative quantification for shotgun proteomic analysis. In particular, we focus on different types of isobaric reagents and their reaction chemistry (e.g., amine-, carbonyl-, and sulfhydryl-reactive). Various factors, such as ratio compression, reporter ion dynamic range, and others, cause an underestimation of changes in relative abundance of proteins across samples, undermining the ability of the isobaric labeling approach to be truly quantitative. These factors that affect quantification and the suggested combinations of experimental design and optimal data acquisition methods to increase the precision and accuracy of the measurements will be discussed. Finally, the extended application of isobaric labeling-based approach in hyperplexing strategy, targeted quantification, and phosphopeptide analysis are also examined.

Comparative Secretome Profiling and Mutant Protein Identification in Metastatic Prostate Cancer Cells by Quantitative Mass Spectrometry-based Proteomics

“…TMT, an isobaric chemical labeling method, is a powerful and convenient tool to estimate the relative abundance of proteins. In particular, isobaric tagging strategy with MSbased proteins is suitable for low-abundance proteins, such as the ones secreted in CM, due to high signal from native peptides (29). We implemented quality check for the proteins, both from CM and from MS-based proteomics with TMT labeling, via western blot and scatter plot, respectively.…”

CONSTANd : A Normalization Method for Isobaric Labeled Spectra by Constrained Optimization

“…Several methods are available to normalize data, such as cyclic LOESS, quantile, and median normalization, etc (43,50). However, when comparing isobaric labeled data from multiple experimental designs, these methods have their shortcomings or require reference samples in each experimental setup to allow for cross-set comparison (51 Therefore, we propose a new and easy to use normalization method, called CONSTANd, which allows accurate normalization of multiplexed, isobaric labeled samples in a datadriven and global manner. Latter terms indicate that all the observed data is used for the normalization procedure.…”

Chemical Proteomic Approaches Targeting Cancer Stem Cells: A Review of Current Literature

“…This technique analyzes relative amounts of cysteine-containing peptides in tryptic digests of paired protein extracts. Isobaric tags for relative and absolute quantitation (iTRAQ) method utilizes a multiplexed isobaric chemical tagging reagent to label primary amines of peptides and proteins (44). iTRAQ reagents usually consist of an Nmethyl piperazine reporter group, a balance group, and an Nhydroxysuccinimide ester group.…”