Absolute Quantitative Analysis (AQUA)

AQUA absolute quantitative analysis is a targeted quantitative proteomics technique widely used in various quantitative proteomics studies. The absolute quantification strategy can be used for the absolute quantification of proteins and their modified states. By using stable isotope-labeled synthetic peptides as internal standards, it can simulate the natural peptides formed by protein hydrolysis, and can also be prepared using covalent modifications (such as phosphorylation, methylation, acetylation, etc.). By using selected reaction monitoring (SRM) analysis in MS/MS, AQUA internal standard peptides are used for precise quantitative determination of the absolute levels of proteins and post-translationally modified proteins after protein hydrolysis. MtoZ Biolabs provides integrate Absolute Quantitative Analysis (AQUA) Service.

The advancement of MS technology has promoted the development of large-scale quantitative analysis methods for intracellular protein expression levels.

Analysis Workflow

Figure 1. Analysis Workflow of AQUA

Protein absolute quantification strategy is an effective detection method for protein quantification and post-translational modifications. Absolute quantification is achieved by using synthetic peptides labeled with stable isotopes: based on discovering the relationship between mass spectrometry signal and protein concentration, the average mass spectrometry signal response of the three strongest trypsin peptides per mole of protein is constant, with a coefficient of variation less than 10%. As long as a given internal standard (a synthetic peptide containing isotopic labels) is provided, the relationship between mass spectrometry signal and protein concentration can be used to determine a universal signal response factor (counts/mol). The universal signal response factors for all quantified proteins are the same.

The absolute quantification method relies on the use of synthetic internal standard peptides, which are introduced into the cell lysate at a known concentration during the digestion process. During the detection process in a tandem mass spectrometer, the hydrolyzed protein samples are analyzed by the SRM method, allowing for direct detection and quantification of natural peptides and isotopically labeled AQUA internal standard peptides. This method's simplicity and sensitivity, combined with the widespread use of tandem mass spectrometers, make the AQUA absolute quantification strategy an effective method for directly measuring protein levels and post-translational modifications from cell lysates.