Absolute Quantification of the Proteome

Proteomics quantitative methods include relative quantification and absolute quantification. Relative quantification proteomics (also known as comparative proteomics) refers to the comparative analysis of protein expression changes in cells, tissues or body fluids under different physiological and pathological conditions. Absolute quantification proteomics is the determination of the absolute amount or concentration of each protein in the proteome of cells, tissues or body fluids.

 

Absolute quantification has clear practical significance for the diagnosis and treatment of clinical diseases. Changes in the quantity of protein biomarkers can directly reflect the development of the disease. Therefore, by detecting the absolute quantity of biomarkers at different stages of the disease, the development of the disease can be judged intuitively. For a long time, absolute quantification of proteins was mainly achieved through immunological methods, such as enzyme-linked immunosorbent assay (ELISA), but this method can only quantify one protein at a time and the types of antibodies are limited. Not every protein can be quantified. With the development of mass spectrometry technology, mass spectrometry-based protein absolute quantification methods have been widely used in absolute quantification analysis of the proteome. Depending on whether there is a label, proteomics absolute quantification analysis methods can be divided into the following two categories:

 

Proteome Absolute Quantification Based on Internal Standard Method

1. Proteome Absolute Quantification by Using Isotopically Labeled Peptides as Internal Standards

In the quantification of proteins, peptides with moderate relative molecular weight (about 0.9~2ku) and strong mass spectrometry signals are selected from the amino acid sequence as templates. Isotope-labeled peptides with the same sequence are synthesized by chemical synthesis or cell culture methods as internal standards. The corresponding protein is absolutely quantified by detecting the ratio of the mass spectrometry signal strength (peak height or peak area ratio) of the peptide.

 

2. Proteome Absolute Quantification by Using Isotopically Labeled Amino Acids as Internal Standards

The protein to be tested is completely hydrolyzed into an amino acid mixture under strong acid conditions. Subsequently, a known amount of isotopically labeled amino acid is added as an internal standard. Using MALDI-TOF-MS mass spectrometry technology, the mass spectrometry peak strength of the amino acid and the internal standard is detected and compared to determine the concentration of the amino acid. Finally, the absolute content of the protein is calculated based on the proportion of this amino acid in the protein.

 

3. Proteome Absolute Quantification by Using Isotopically Labeled Peptides for Labeling

A pair of isotopic labels are respectively bound to the peptide to be tested and the synthetically identical amino acid sequence peptide, forming a pair of isotopically labelled compounds with the same physical and chemical properties but different relative molecular weights. The absolute amount of protein or peptide in the sample can be determined by mass spectrometry.

 

4. Proteome Absolute Quantification Based on Inductively Coupled Plasma Mass Spectrometry for Element Determination

A suitable phosphorylation compound BNPP is added as an internal standard in the mixture of phosphopeptides after enzymatic digestion. After HPLC separation, the mass spectrometry peak intensity of phosphorus element is detected in ICP-MS. At the same time, the amino acid sequence of peptide is qualitatively identified by ESI-MS under the same chromatographic conditions. Based on the peak intensity of the internal standard and the known added amount, as well as the number of phosphate residues contained in each peptide, the absolute amount of phosphopeptides can be calculated.

 

Label-Free Proteome Absolute Quantification

The amount of protein is positively correlated with the signal of its enzymatic peptides in mass spectrometry. By adding a known amount of a standard protein (non-test protein) to the test sample, and then comparing the signal intensity of the added standard protein's peptides with the protein's peptides in the test sample in mass spectrometry, and finally combining the amount of added standard protein, the absolute content of protein in the test sample can be calculated.

 

MtoZ Biolabs uses Thermo Fisher's Q ExactiveHF mass spectrometry platform combined with Nano-LC chromatography to provide fast and efficient protein absolute quantification service technology package. You only need to tell us your experimental purpose and send your cells to us, we will be responsible for all subsequent matters of the project, including cell culture, cell labeling, protein extraction, protein enzymatic digestion, peptide separation, mass spectrometry analysis, mass spectrometry raw data analysis, bioinformatics analysis, welcome to consult for free!