Techniques for Quantitative Proteomics

With the development and maturity of mass spectrometry, the technical advantages of high throughput, high sensitivity and wider dynamic range have been emerged, which provides a reliable research method for proteomics research. In recent years, quantitative proteomics has become one of the research hotspots in omics field.

Quantitative proteomics is an emerging technology used for accurate quantification and identification of proteins expression in biological samples. It can not only identify the proteins expressed in different states, but also accurately quantify their abundance. According to different quantitative purposes, quantitative proteomics can be divided into two categories relative quantitative proteomics and absolute quantitative proteomics. Relative quantitative proteomics, also known as comparative proteomics, is a comparative analysis of protein expression level in cells, tissues or organism in different physiological or pathological conditions. Absolute quantitative proteomics is the determination of the absolute quantity or concentration of each protein in a proteome.

Four Methods Commonly Used in Quantitative Proteomics Research

Label-free

 

Label free is a method in quantitative proteomics that aims to analyze the relative differences between different tissues or cells in protein. However, unlike other quantitative proteomics methods, label free quantitative proteomics does not require an expensive stable isotope label as an internal standard, but just need to analyze the mass spectrum data and compare the signal intensity of the corresponding peptide segment, thus obtaining the expression level of protein between two or among more biological samples. Compared with isotopelabeling methods, label-free is simple to operate and can be used to quantify total protein variance in any sample. But there are still some limitations that it puts high requirements for stability of the experimental operation and can only analyze one sample each time.

SILAC


Stable isotope labeling by amino acids in cell culture (SILAC) is a simple and accurate method in mass spectrometry-based quantitative proteomics, for the in vivo incorporation of specific amino acids into all mammalian proteins. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. SILAC removes false positives in protein-interaction studies, and reveals large-scale kinetics of proteomes. Besides that as a quantitative phosphoproteomics technology, SILAC directly uncovers important points in the signaling pathways that control cellular decisions. SILAC provides accurate relative quantification without any chemical derivatization or manipulation and enables development of elegant functional assays in proteomics.

 

iTRAQ

 

Isobaric tags for relative and absolute quantitation（iTRAQ）is a new LC-based technology which can label peptide segments obtained from protein hydrolysis. It allows simultaneous identification and quantitation of proteins in four or eight different samples using tandem mass spectrometry, and also can detect lower abundance proteins. It is especially suitable for differential protein analysis using multiple treatments or samples from multiple processing times. It has been widely applied to comprehensive study of the protein quantitative, including the quantitative analysis of post-translational protein modification, the qualitative and quantitative analysis protein and membrane protein obtained by affinity purified samples.

 

MRM

 

Multiple reaction monitoring (MRM) is a method of targeting quantitative proteomics, which based on target molecular information, and the acquisition of mass spectrometry information of the selected data. MRM is capable of rapid, sensitive, and specific quantitation analysis in highly complex sample matrices, and also can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. Recently, MRM assay approach has been applied to the measurement of specific peptides in complex mixtures such as tryptic digests of plasma.

Applications and Significance of Quantitative Proteomics

Quantitative proteomics technology has been widely applied to identify diagnostic expression patterns specific to pathological cellular states, and to provide clues to the genes and gene products that are responsible for, or correlate with, a specific state of a system. It also has been used to accurately quantify human tumor proteomes, and to investigate the mechanism of the antifungal agent and the target of drug action.

Quantitative proteomic technology not only is of significant interest in studies aimed at discovering disease biomarkers and providing new insights into biological pathways, but also holds significant promise for the discovery of diagnostic or prognostic protein markers for the detection of new therapeutic targets. 

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