Six Techniques That Make Your Proteomics Research Easier (Part II)

SILAC quantification is suitable for the analysis of living cultured cells and can be used for the identification and quantification of whole protein and membrane proteins. SILAC is mainly quantified by MS1, and iTRAQ/TMT is mainly quantified by MS2. There is not much difference in quantitative accuracy and protein resolution between the two, but due to the complicated operation, long time span and high cost required by SILAC technology implementation, it has not been widely commercialized.


Label-free, as its name implies, is a method for the identification and quantification of protein enzymatic peptides by non-labeled quantification. It is not necessary to use a reagent containing a stable isotope label. It is only necessary to compare the intensity of MS1 signal between the two groups of samples in a specific peptide to obtain a change in the amount of protein expression between the samples, which is usually used for the analysis of mass spectral data produced by large-scale protein identification and quantification.

Label-free technology is simple to operate and requires fewer samples. It can be used to quantify total protein differences in any sample. A single test can identify and quantify more proteins. Therefore, Label-free technology is suitable for quantitative comparison of large sample sizes, as well as experimental designs that cannot be quantified by labeling. However, the requirement for stability and repeatability of the experimental operation is comparatively high, and the accuracy is also poorer than that of Labeling quantitation which depends on the stability of the mass spectrometer.

The current popular proteomics research methods, such as iTRAQ/TMT, Label-free and SILAC, are all rely on DDA (data-dependent acquisition) technology. Digest protein samples into peptides, ionize and analyze by mass spectrometry. MS2 can only collect the top 20 information of MS1 signal, so it will cause information leakage. In the process of parent ion selection, the mass spectrum is more inclined to scan the peptide with high abundance.

In response to the above problems, a new mass spectrometry technology SWATH (Sequential Windowed Acquisition of all Theoretical fragment ions) came into being. SWATH is a DIA (data-independent acquisition) technology. Its concept is to fragment all the parent ions in a specific mass range, collect the fragment ions of all the parent ions, and quickly and sequentially scan all fragment ions in the wide opening of the adjacent parent ion.

DIA quantification is different compared with traditional DDA quantification. DIA has the advantages of high sensitivity, high reproducibility, high quantitative accuracy and large throughput. SWATH technology is mainly used for proteomic quantification, protein complex identification and host protein analysis.

There are two main types of techniques for targeted proteomics, MRM (multi reaction monitoring) and PRM (parallel reaction monitoring). SRM (selective reaction monitoring) is the gold standard for proteomic targeting. The principle of MRM and SRM is the same, which is equivalent to doing multiple sets of SRM at the same time, and is mainly relying on the triple quadrupole mass spectrometer. After detecting the parent ion which is specific to the target analysis, the selected specific parent ion is subjected to collision induction to remove the interference of other daughter ions, and only the selected specific ions are collected for mass spectrometry signals to achieve the purpose of getting a more specific, sensitive, and accurate analysis on the target.

PRM is an upgraded version of SRM-based technology that relies heavily on high-resolution Orbitrap mass spectrometers. Thanks to the advantages of a high-resolution and high-quality precision analyzer, the PRM achieves a full scan of the daughter ions, eliminating the need to select the parent ion fragments. Compared to SRM/MRM, the operation of PRM is simpler, the selectivity and sensitivity of targeted quantitative results are higher, the reproducibility is better, and the anti-interference ability is stronger in complex backgrounds.

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