Techniques Commonly Used in Quantitative Proteomics

With the development of proteomics and biomass spectrometry, large scale quantitative proteomics has become an important aspectof proteomics. Quantitative proteomics plays important roles in deciphering the biological progress, understanding the development of the disease, and discovering as well as verifying biomarkers for the diagnosis and therapy of diseases. The common quantitative proteomics services are label free and stable isotope labeled quantification, eg. SILAC, iTRAQ.

Quantitative proteomics based on Label Free

Label free quantification is a method in mass spectrometry that aims to determine the relative amount of proteins in two or more biological samples.

Label-free approaches to quantitative proteomics have gained prominence in recent years since no additional chemistry or sample preparation steps are required. Methods: comparing the ratios of integrated peak areas/intensities of phosphorylated to nonphosphorylated peptide ions between two samples, a strategy to assess the stoichiometry of phosphorylation sites. And the popular method of quantifying protein levels from different samples based on the number of MS/MS spectra that identify the protein of interest (spectral counting).

Quantitative proteomics based on TMT/iTRAQ

Initial commercial isobaric tag offerings isobaric tags for relative absolute quantitation (iTRAQ) in 4-plex and 8-plex configurations and tandem mass tags (TMT) in a 6-plex configuration. Recently, TMT reagens were expanded to 10-plex configuration, by the addition of mass defect-based variants , for use with high-resolution MS platforms. These products have established themselves as powerful tools for high-throughput quantitative proteomics studies.

iTRAQ has been uitilized to identify biomakers for various disease condition. It has been suggested to be suitable for the discovery of biomakers in a wide range of body fluids and tissues, including serum and plasma.

Quantitative proteomics based on SILAC/Dimethyl labeling

Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is a technique based on mass spectrometry that detects differences in protein abundance among samples. It is a popular method for quantitative proteomics. SILAC and chemical labeling by stable isotope dimethyl labeling have comparable accuracy and quantitative dynamic range in unfractionated proteome analyses and affinity pull-down experiments. Stable isotope dimethyl labeling is a chemical labeling method for quantitative proteomics; it labels the N-terminus of proteins/peptides and at labeling methods, stable isotope dimethyl labeling is applicable to a wide variety of samples. It was recently used in a large-scale phosphoproteomics study from mouse liver tissues. The methods of SILAC and dimethylis are basically consistent, which are widely used in quantitative proteomics analysis.

SILAC-based CoIP-MS analysis

Co-immunoprecipitation (co-IP) is a popular technique to identify physiologically relevant protein–protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein. SILAC-based CoIP-MS has emerged as a very powerful method to study cell signaling, post translation modifications such as phosphorylation protein–protein interaction and regulation of gene expression[,]. In addition, SILAC-based CoIP-MS has become an important method in secretomics, the global study of secreted proteins and secretory pathways.

Quantitative proteomics based on MRM

Multiple Reaction Monitoring (MRM), also known as selective reaction monitoring, employs targeted mass spectrometry (MS)-based data acquisition by monitoring prespecified peptide precursor ions along with a defined handful of their fragments for highly sensitive and specific quantification. Due to its high sensitivity, accuracy, and reproducibility, MRM has become increasingly used for targeted peptide and protein quantification and has been successfully used in several high-throughout clinical applications. A key aspect of MRM assays is that each protein has multiple, semi-independent, layered observations which collectively indicate protein quantity.

Quantitative proteomics based on SWATH

Sequential Window Acquisition of all Theoretical fragment ions (SWATH) is an emerging technology in the field of mass spectrometry based proteomics. In a SWATH experiment, proteins are digested and either directly infused or, more often, separated by liquid chromatography prior to analysis on a Triple TOF mass spectrometers, a Q-Exactive mass spectrometer, or any instrument with sufficiently high scan speed and a quadrupole mass filter.

The quantitative information of the proteome can help us understand the biological function of proteins in the interaction network. Quantitative proteomics has been widely used in screening biomarkers in various cancers and is becoming an important tool for differential and dynamic proteomics research. With the determination of the absolute quantitation of the biomarker, we can directly judge the status of a disease, which will benefit the clinical diagnosis and the treatment of the disease.







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