What are the advantages of iTRAQ/TMT proteomics analysis?
Answer: The goal of proteome comparison between different samples is achieved by means of actual labeling of samples from different sources with different molecular weights.
- Complete qualitative and quantitative analysis of all samples (iTRAQ 8/TMT 16) in one go
- Reduces experimental error
- Eliminates inter-sample variation caused by instrumentation
- High stability
- Simple operation
- Simple data analysis process
- Suitable for quantitative analysis of proteins from all species
What is the main difference between iTRAQ/TMT?
Answer: TMT (Tandem Mass Tag) and iTRAQ (isobaric tags for relative and absolute quantitation) technologies are in vitro peptide labeling and quantification technologies developed by Thermo and AB SCIEX, respectively. They are the two most widely used tagging technologies for quantitative proteomics and have similar principles. The main difference is that TMT can label up to 16 different groups of biological samples and iTRAQ can label up to 8 different groups of biological samples simultaneously.
What do I need to pay attention to when performing iTRAQ&TMT labeling experiments. What are the requirements for samples?
Answer: iTRAQ&TMT is suitable for relative quantitative analysis of the same tissues, and cannot be identified with or without. Different tissue parts cannot be labeled together on the machine, nor can samples from unused species be labeled together on the machine. The total protein requirement is higher than LFQ, 100ug or more.
Why can't iTRAQ/TMT be used for species or samples with large differences?
Answer: This is mainly due to the technical characteristics of iTRAQ/TMT. Data analysis for marker quantification are based on shared proteins. The specific proteins will be filtered out and cannot be analyzed in the later data. Therefore, iTRAQ/TMT cannot be used for species or samples with large differences.
Can samples of different tissue types be used in the same batch for iTRAQ/TMT?
Answer: The samples must be of the same species and the same tissue type in order to be analyzed in the same batch. If you select samples of different sample types, their specific proteins will be filtered out.
Why can't some of the iTRAQ/TMT samples from the same batch be selected for individual analysis?
Answer: iTRAQ/TMT is a mixed batch and the data quality control is performed on a run basis (i.e., the starting fraction). The total protein obtained is the common protein of all samples on the machine. So there is no way to analyze it separately.
Is it possible to look for the presence or absence of differential proteins or unique proteins between different samples by iTRAQ/TMT experiments?
Answer: No, it is not possible. There are several reasons for this.
- The results of the iTRAQ experiment use Proteome Discoverer software to search the library, and the algorithm analysis of this software assigns values for both very large and very small values.
- For protein quantification, the software uses a combined peptide level assay, which makes it difficult to exclude other similar ion interferences.
- A small number of proteins with no quantification values may be the result of not detecting a unique peptide, or the reporting ion peak is too low and does not indicate the absence of the protein.
How to select the differential proteins screened by iTRAQ/TMT experiments for validation?
Answer: Large-scale histology data require specific validation experiments at a later stage to support the credibility of the histology data and conclusions. Selection can be done in the following ways.
- Study relevant literature reports of proteins known to be closely related to the research topic for further study
- Preferentially selecting proteins with large quantitative proteomic difference multiples for study
- Selection of proteins with relatively high number of unique peptides among the identified differential proteins (the higher the number of unique peptides, the higher the confidence level)
- Selection of proteins with good intergroup parallelism and consistent expression trends for further study (the more consistent the trend, the more plausible the protein change)
- Combine with functional analysis or pathway analysis (bioinformatics analysis) to select meaningful proteins for study
How can the differential proteins screened by iTRAQ/TMT experiments be further validated?
Answer: There are many validation methods, such as Western blot, ELISA , PRM (parallel reaction monitoring), which is based on targeted proteomics to study the expression changes of specific proteins. It can also be validated with each other using metabolomics, genomics and other multi-omics.
Why the differential proteins screened by iTRAQ/TMT experiments are not validated subsequently?
Answer: It can be considered from the following aspects.
- whether the selection of validation target is reasonable, whether the experiment is reproducible and reliable
- Whether the validation of protein modification level is time-sensitive
- There may be expression differences at different stages of validation such as gene level to protein level. The screening of large scale data will have some false positives.
Why are proteins detected by Elisa and WB not detected in iTRAQ/TMT or protein identification assays?
Answer: Western blot assays amplify the target protein signal by many steps and are highly sensitive, (except for specific binding) specific, and detect the target protein specifically. It is virtually unaffected by the abundance of background proteins in complex samples. For pure protein samples, mass spectrometry detection is sensitive up to the fmol level. However, in complex samples, mass spectrometry is not specific for detection, but selects the proteins with higher abundance in the sample for preferential detection. In contrast, proteins with lower abundance are not detected because the peptide signal is too weak to be swamped. Therefore, if the target protein to be detected in the sample is in low abundance (or has a small molecular weight), even if WB can detect it, mass spectrometry does not necessarily detect it.
