Quantitative analysis of protein
Why do we study protein quantification? What's the use of protein quantification?
To evaluate the biological function and modification of proteins, to understand the signal transduction mechanism and disease-specific biomarkers in cells, it is of great significance to study the changes of proteome under different experimental conditions. The emerging techniques of quantitative proteomics based on mass spectrometry provide a powerful tool for quantifying and systematically evaluating the quantitative differences in the protein spectra of different samples, and are increasingly becoming an important part of biomedical and clinical research.
Discovery-based quantitative proteomics compares diseased and normal samples of proteomics globally and has been used extensively in the study of various human diseases with the aim of identifying biomarkers and/or revealing disease pathogenesis. Methods for obtaining quantitative proteomic data are being developed with very precise stable isotope labeling (SIL) and unlabeled methods.
How to study protein quantification?
Here are some techniques for studying protein quantification:
Absolute quantification is a kind of targeted quantitative proteomics technology, which has shown strong efficacy and is increasingly used in various quantitative proteomics researches. Absolute quantification is based on finding an unexpected relationship between MS signal response and protein concentration: the average MS signal response of the three strongest trypsin peptides per mole of protein is less than or equal to 10%.Given an internal standard, this relationship is used to calculate a common signal response factor. The generic signal response factor (count /mol) shown is the same for all tested proteins. Although isotopic methods only establish relative quantification of expressed proteins, absolute quantification (AQUA) strategies can provide information for accurate determination of protein expression and post-translational modification levels. AQUA relies on the use of synthetic internal standard peptides, which are introduced into cell lysates at known concentrations during digestion. In a tandem mass spectrometer, AQUA labeled natural peptides and isotopic markers can be directly detected and quantified by selecting reaction monitoring (SRM) assay for proteolytic samples. The simplicity and sensitivity of the method, combined with the widespread availability of a tandem mass spectrometer, make the AQUA strategy a highly useful method for measuring protein levels and post-translational modifications directly from cell lysates.
Silac-based quantitative proteomics (SILAQ) is an innovative high-throughput quantitative analysis technique for large protein complexes, protein-protein and protein-small molecular interactions. SILAC can reveal how inhibitors or other disturbances specifically affect the dynamic properties of proteins and cell distribution. It can also be used as a sensitive and effective way to identify specific interacting partners of proteins in cells.
SILAC relies on the incorporation of a given "light" or "heavy" form of amino acid metabolism into two samples. Since the amino acids of the two isotopic markers are basically the same in chemistry, their binding does not interfere with normal cell growth, leading to protein/peptide being distinguished by mass, and thus very suitable for mass spectrometry analysis. The method relies on the binding of amino acids to the replaced stable isotope nuclei. Thus, in one experiment, two groups of cells grew in the same medium, only one containing a specific amino acid in the "light" form and the other containing a specific amino acid in the "heavy" form (e.g., l-lysine labelled 12C and 13C, respectively).Then enzyme digestion and LC/MS analysis were performed on SILAC samples. Protein quantification is done at the protein level. In addition, SILAC method is very suitable for monitoring changes of post-translational modification. Examples of these applications include measuring changes in protein phosphorylation and methylation.
ITRAQ protein quantification is suitable for unbiased and aimless biomarker discovery. The relative quantification of proteins for biomarker discovery in complex mixtures can be achieved easily and quickly by using iTRAQ technology. ITRAQ is well suited for comparison of normal, disease, and drug therapy samples, time series studies, biological replication, and the provision of relative quantification.
Samples were prepared under different treatment conditions and then cell lysis was performed to extract the protein. After estimating the protein concentration of each sample using standard protein assay, enzymes (such as trypsin) are used to digest the protein to produce proteolytic peptides. Each peptide digestion was labeled with a different ITRAQ reagent, and the labeled peptides were then combined into a sample mixture. The combined peptide mixture was analyzed using lc-ms /MS for identification and quantification. Database searches are then performed using fragment data to identify labeled peptides and hence the corresponding proteins. The tagged fragments produce low molecular mass reporter ions which can be used in relative quantification of peptides and proteins of their origin.
TMT quantification was performed by measuring the intensity of ions released from the tags in tandem MS mode (MS2) during peptide fragmentation. Select the precursor ions in the full scan mode (MS1) to be fragmented. Since the ion selection step reduces the noise level, it is beneficial. Ideally, only one of the selected precursors in the precursors fragment is crushed. However, it is common for other precursor ions to be trapped in the specified m/z window and broken in practice with the selected precursor. This is a common separation or mixing of precursors. Isostatic markers work best for mass spectrometers allowing MS3 level quantification, such as the fusion orbital of THEMO. Additional filtering steps allow almost complete correction of CO isolation caused by MS1 precursor contamination.
After the above contents, we’ve learned a lot about quantitative proteomics, and those who are interested can continue to explore the quantitative analysis of protein on this basis, and you may be the next person to discover the new technology!
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