Mass Spectrometry-Based Proteomics Quantification: iTRAQ
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This document discusses iTRAQ (isobaric tag for relative and absolute quantitation), a method for determining the amount of proteins from different sources in a single experiment. It describes the basic structure of iTRAQ reagents, which consist of a unique reporter group, peptide reactive group, and neutral balance group. The principle and workflow of iTRAQ is explained, involving labeling samples with iTRAQ tags, combining samples, performing MS/MS for identification and quantitation. Factors affecting iTRAQ results and its advantages/disadvantages are briefly covered. An example application of iTRAQ to identify tyrosine phosphorylation sites is provided.
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Mass Spectrometry-Based Proteomics Quantification: iTRAQ
- 1. iTRAQ-based Proteomics Approaches Presented by Creative Proteomics
- 2. Introduction iTRAQ was developed by Applied Biosystems Incorporation in 2004. iTRAQ is a isobaric labeling method to determine the amount of proteins from different sources in just one single experiment. 1 2 3 iTRAQ is an acronym of Isobaric tag for relative and absolute quantitation.
- 3. The structure of iTRAQ reagents • The isobaric tagging reagents consist of a unique charged reporter group, a peptide reactive group, and a neutral balance group. • The peptide reactive group covalently links an iTRAQ Reagent isobaric tag with each lysine side chain and N-terminus group of a peptide, labeling all peptides in a given sample digest. • The neutral balance group ensures the iTRAQ labeled-peptide displays the same mass to maintain an overall mass of 145 Da for 4-plex and 305 Da for 8-plex. The basic structure of iTRAQ reagent-4 plex and 8 plex The Principle and Workflow of iTRAQ N N N 114 31 Peptide 115 30 Peptide 116 29 Peptide 117 28 Peptide Reporter group Balance group Amine specific peptide reactive group(NHS) Total mass: 145 N N N 113 192 Peptide 114 191 Peptide 115 190 Peptide 116 189 Peptide 117 188 Peptide 118 187 Peptide 119 186 Peptide 121 184 Peptide Reporter group Balance group Amine specific peptide reactive group(NHS) Total mass: 305 iTRAQ reagent 4-plx iTRAQ reagent 8-plx MS fragment site
- 4. Principle of iTRAQ-iTRAQ reagent 4-plex as an example Mix Fragment MS m/z 117 116 115114 m/z LC/MS 114 31 115 30 116 29 117 28 Total mass: 145 iTRAQ labelN N N Peptides Software Analysis
- 5. Control sample Test sample7Test sample1 Reduce, cysteine block and digest Label with iTRAQ Combine the iTRAQ reagent labeled control and treatment sample digests into one sample mixture Clean up perform high-resolution fractionation Analyze the mixture by MS/MS for protein identification and quantitation …… control treatment Reduce, cysteine block and digest Label with iTRAQ Reduce, cysteine block and digest Label with iTRAQ Lysis Lysis Lysis Workflow of iTRAQ
- 6. Factors affecting results of iTRAQ • Evaluation of labeling efficiency and isotope impurity correction • Ratio compression and its correction • Reporter ion intensity dynamic range • Effect of unique and shared peptides in inferring protein ratios • Estimation of protein fold changes • Comparison of multiple isobaric labeling experiments
- 7. Advantages and disadvantages Advantages Disadvantages Using mass spectrometry Costly Ability to analyze proteins from cell, tissues or serum Very sensitive to contamination from salts Multiplexing ability A requirement of sophisticated software Reduce overall time and variation Variability arising due to the inefficient enzymatic digestion
- 8. iTRAQ used to identify and quantify tyrosine phosphorylation sites upon insulin stimulation. An example Schmelzle K, Kane S, Gridley S, et al. Temporal dynamics of tyrosine phosphorylation in insulin signaling. Diabetes, 2006, 55(8): 2171-2179.
- 9. iTRAQ at Creative Proteomics Cutting-edge facilities Professional staff Reliable results • iTRAQ-based proteomics analysis service • TMT-based proteomics analysis service • SILAC-based proteomics analysis service • Absolute quantification (AQUA) service • Label-free quantification service • Semi-quantitative proteomics analysis service
Editor's Notes
- iTRAQ (Isobaric tag for relative and absolute quantitation) was developed by Applied Biosystems Incorporation in 2004. iTRAQ is a isobaric labeling method to determine the amount of proteins from different sources in just one single experiment. The iTRAQ reagents available always are set of 4-plex and 8-plex mass tages that can be used to label and et quantitative information on up to 4 and 8 different samples in one experiment
- A mixture of peptides is obtained by hydrolyzing the protein sample. All peptides in the sample are labeled with different iTRAQ reagents, and all labeled protein samples are mixed. The peptides are subjected to tandem mass spectrometry to obtain the mass spectrum. In the first-stage mass spectrometry, after being labeled with different isotopes regardless of which iTRAQ reagent is used, the same peptide from different sources is completely identical in molecular weight and they appeared as the same peak. Samples of the same peak are collected to perform a second-stage mass spectrometry, in which the bond between the balance group and the peptide reactive group is broken and the balance group is lost. The same peptide with different isotopic labels produces different masses of reporter ions, and the reporter ions exhibit different peaks. Quantitative information of the same peptides between different samples can be obtained by analyzing related data with software and databases.
- In the general workflow for an iTRAQ experiment, each sample is reduced, cysteine blocked, and digested with trypsin. Each sample is labeled with a different iTRAQ tag in a single tube. And then we can combine all iTRAQ reagent-labeled samples into one sample mixture for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
- Quantitative proteomics using mass spectrometry-based approach is one of the most important advantages of i TRAQ, especially in the field of clinical proteomics. It has the ability to analyze proteins from various source of samples, including cells, tissues, or serum. Multiplexing ability is another great advantage of i TRAQ. There are eight reporter ions available that therefore achieve multiplexing at the level of eight samples. Also, i TRAQ can reduce overall time and variation. On the other hand, iTRAQ reagents are extremely costly and also extremely sensitive to contamination from salts. Sophisticated software is required for analyzing iTRAQ data. Moreover, the variability arises due to the inefficient enzymatic digestion.
- There is an example that iTRAQ was used to identify and quantify tyrosine phosphorylation sites upon insulin stimulation. In this experiment, adipocytes were stimulated with insulin for different times. After cell lysis and digestion, the researchers used iTRAQ to label resulting peptides. The labeled peptides were combined for further analysis (phosphotyrosine peptide immunoprecipitation, immobilized metal affinity chromatography, and LC-MS/MS). After LC-MS/MS, the identity of the peptide was determined and the area of the tag masses 114, 115, 116, and 117 mass-to-charge ration (m/z) was used to calculate the ratio of phosphorylated peptides at the different time points compared with 5-min insulin stimulation.