Q&As About Proteomics (Q6-Q10)

Former Q&As can be read at Q&As About Proteomics (Q6-Q10).

Q6. What software is used for mass spectrometry analysis for the raw data of iTRAQ? How to judge the differential protein?

We use the proteinpilot 5.0v with the Triple TOF 5600 plus. There are a large number of proteome-related literature published using this software. It has been widely used as a proteomic identification and quantification software. Currently, there is no uniform standards for the conditions of differential protein screening. In actual screening process, we need to combine the specific experimental results to make flexible changes. Try to control the total number of differential proteins in each group to be about 200 or about 10% of the total protein number. It is more conducive to our subsequent bioinformatics analysis. General credible protein screening can be screened according to FDR, unused and unique peptides. For the criteria of quantifying differential proteins, screening is generally performed according to values such as Flod change and P-value.

Q7. Can a species without whole-genome sequencing be a proteome?

At present, genome-wide sequencing of a large number of species have been completed. Their corresponding genes have also been predicted. Unsequenced species can be used for proteome research through the genetic sequence of related species. If there are no good related species, a slightly larger genus or family database can be used to research. In addition, if the researchers have done transcriptome, they can also use relevant data to build a local library as a mass spectrometry database for proteins.

Q8. Why do the proteins detectable with elisa and WB fail to be detected in iTRAQ experiment?

Firstly, there are tens of thousands of proteins in human plasma. It is often the case that only five to six hundred of them can be detected by mass spectrum, which indicates that our method is OK. Secondly, ELISA/WB is relatively sensitive to mass spectrometry. The former has a magnification effect, because of the use of corresponding antibody. Therefore, the protein of interest will be detected once proper antibody is selected. In general, it is normal that proteins detected by ELISA/WB are not detected in iTRAQ experiment.

Q9. Two differential proteins from iTRAQ result were verified with elisa and found to be negative. Why? What is the probability of being negative? Will we mention this probability generally?

In iTRAQ results, whether the selected protein scores high enough, the fold change is great, or the specificity of selected antibody performs well may all affect the results of WB and iTRAQ. If there is no problem exists in the above three aspects, the probability of being positive will be around 80%.

Q10. What mass spectrometers are used in iTRAQ, TMT, and Label Free experiments? Is their quantification based on MS or MS/MS?

The experimental technology based on LC-MS/MS mainly uses ABI5600-Triple-TOF, and the company also has a Thermo Q-Exactive mass spectrometer platform. Some experimental projects use QE-Exactive. The label quantification techniques, iTRAQ and TMT, are quantified based on the peak of the reporter ion of MS/MS, while Label Free is quantified based on the peak of MS.

Prime Jones is a senior researcher from MtoZ Biolabs. She is specialized in the field of proteomics study.