Differences Among Five Commonly 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.

Label-free

Label Free Workflow
Label free quantitative proteomics does not require specific labeling of comparative samples. It is only necessary to compare the chromatographic mass-spectrometry response signals of specific protein peptides between different samples, so as to obtain the changes in the expression of proteins between samples. It is commonly used to analyze mass spectral data generated during the identification and quantification of large-scale proteins.

iTRAQ

iTRAQ Workflow
Isobaric tags for relative and absolute quantification (iTRAQ) technology is an in vitro isotopically labeled relative and absolute quantification technique developed by AB SCIEX. This technology uses multiple isotope reagents to label the N terminus or lysine side-chain groups of protein peptides. Through high-performance mass spectrometer tandem analysis, it can simultaneously compare protein expression levels between up to 8 samples. It is a commonly used high-throughput screening technique for quantitative proteomics in recent years.

SILAC

SILAC Workflow
SILAC refers to Stable Isotope Labeling By Amino Acids In Cell Culture. There are three experimental procedures. Firstly, add light, medium or heavy stable isotopically labeled essential amino acid (lysine and arginine), enabling newly synthesized proteins to carry stable isotope labels through the normal metabolism of cells.  Secondly, Mix equal amount of protein and analyze by mass spectrometry after enzymatic hydrolysis. Then, perform relative quantification by comparing the area size of isotope peaks in the first-order mass spectra, while sequencing the peptides with secondary spectrum for protein identification.

MRM

MRM Workflow
MRM is a kind of data acquisition method that sets mass spectrometry detection rules based on known information or hypothetical information and records ions in accordance with the rules to remove a large amount of interference which does not conform to regular ion signals. The key is to be able to detect the specific precursor ions first, and then choose selected specific precursor ions only to collision induction, so as to remove the interference of other daughter ions, and only the selected specific ions are collected for mass spectrometry signals.

SWATH

SWATH Workflow
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.

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.

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