Comparison of Five Common Used Quantitative Proteomics Analysis Methods
The proteome refers to the full set of proteins expressed by an organism. Proteomics essentially refers to studying the characteristics of proteins on a large scale, including the expression level of proteins, post translational modification, protein-protein interactions, etc., thereby obtaining overall and comprehensive understanding of disease occurrence, cell metabolism and other processes at protein level. The emergence and development of biological mass spectrometry provide a reliable and dynamic range of research tools for quantitative proteomics analysis.
Currently, there are five mainstream quantitative proteomics methods, label-free, iTRAQ, SILAC, MRM (MRMHR), and SWATH.
iTRAQ
iTRAQ quantitation does not rely on samples, and can detect low abundance proteins with accurate quantification. Eight samples can be analyzed at the same time, which is suitable for studying the difference of protein expression levels in tissue samples under different pathological conditions or at different developmental stages.
Label-free
Label-free quantification method is easy to operate and can perform total protein differential quantification for any sample. However, it requires high stability and repeatability of experimental operations, and the accuracy is also worse than that of markers. Therefore, Label-free technology is suitable for quantitative comparisons of large sample sizes, as well as experimental designs that cannot be quantitatively achieved with markers.
SILAC
Silac is an in vivo labeling technology that is closer to the real state of the sample, and has a stable labeling efficiency of up to 100%. It is suitable for whole-cell protein analysis and the identification and quantification of membrane proteins. Each sample requires only tens of micrograms of protein.
SWATH
SWATH is a new mass spectrometry acquisition mode technology jointly developed by Dr. Ruedi Aebersold of the Swiss Federal Institute of Technology in Zurich, Switzerland, and his team along with AB-SCIEX in 2012. It is an extension of the MS/MSALL technology. Compared with the traditional shot-gun technology, the SWATH acquisition mode can scan all the peptide precursor ions in the scanning interval and perform secondary fragmentation to obtain complete peptide information. It is a truly panoramic view, high-throughput mass spectrometry technology.
MRM
The MRM technology eliminates a large number of interfering ions through two-stage ion selection, reducing the chemical background of the mass spectrometer, and significantly improving the signal-to-noise ratio of the target analytes, thereby achieve high sensitivity of detection, good reproducibility, high accuracy, etc. The difference in protein expression levels suitable for known protein sequences can be used to detect less abundant proteins, but only about 20 target proteins can be detected in a single MRM assay.
How to Choose a Suitable Quantitative Proteomics Analysis Method?
iTRAQ quantitative proteomics is a popular method, which does not rely on samples. It can do different quantification of total protein in any sample, and is quantitatively accurate. Although label-free does not limit the sample either, the quantitative accuracy is difficult to guarantee. SILAC is a proteome quantification at the cell level, but the quantification of tissue is difficult, and SILAC is expensive to cultivate and is not suitable for commercialization. Both MRM and SWATH are target proteome related quantitative models, but SWATH can perform the quantification of thousands of proteins with extremely high accuracy and fluxes much higher than MRM for subcellular structures, bacteria, fungi, cell secretions, etc. SWATH works very well, but SWATH is relatively expensive.