Over the years, the absolute quantification (AQUA), based on isotope-labeled peptides has become a resultful means of quantifying proteins. AQUA can quantitatively study various complicated biological samples and provide valuable new tools for proteomics.

Properties of AQUA Peptides
The target peptides of the protein to be quantified should be able to ionize effectively and be easily detected by mass spectrometry (MS).
The target peptides should avoid containing methionine and cysteine and other sites that are prone to chemical modification.
The target peptides should not contain special sequences such as the aspartic acid-glycine that is easily broken by enzymatic cleavage.
The target peptides should avoid bearing or near post-translational modification sites.
The length of the peptide should be controlled within 7-20 amino acids as much as possible, in order to obtain a better MS signal.
Experimental Method
The hydrolyzed peptides of the target protein are obtained by experimental or predictive methods. A certain amount of isotopic label was added to the analysis sample as a reference to reduce quantitative differences caused by matrix effects, ionization efficiency and unstable instrument response signals during the experiment. The isotope-labeled peptides are incorporated into cell lysates at known concentrations by proteolysis. The isotope-labeled peptide and the tested peptide show the same physical and chemical properties and different mass-to-charge ratios. Therefore, the target protein of the sample is calculated by the ratio of the labeled/unlabeled peptide and the absolute amount of the labeled peptide.

AQUA technology can accurately and sensitively detect proteins with medium and high abundance in various samples and under various conditions, and requires trace amounts of samples. AQUA peptides can be incorporated proportionally when quantifying a set of proteins with a wide dynamic range. AQUA peptides are readily available and simple to use, enabling comparability of quantitative data from different laboratories and different instruments. Besides, AQUA can also be applied for absolute quantification of post-translationally modified proteins, such as phosphorylation modification, using each of the unmodified/phosphorylated AQUA peptides as the reference to quantify the total protein expression and the occurrence of phosphorylation protein abundance.

Technology applications
Through the high-throughput screening of proteomics, combined with AQUA-based and Multiple Reaction Monitoring (MRM) / Parallel Reaction Monitoring (PRM)/ Selected Reaction Monitoring (SRM) technology to verify in large-scale samples, it is found that the diagnostic markers of protein diseases are better than the traditional alpha-fetoprotein (AFP) and glycoproteins. The combination of markers (Apolipoprotein H, Orosomucoid 2 (ORM 2), and AFP was more accurate in the diagnosis of liver cancer. The mass spectrometry-based AQUA strategy has also been used in subunit studies of protein complexes to detect the levels of different subunits in serum. The AQUA strategy can also be utilized for quantifying low-abundance proteins involved in gene silencing and quantitatively identifying the cell cycle-dependent phosphorylation of proteins. Furthermore, AQUA is also applicable to the external standard curve method. Compared with the traditional single-point quantitative method, the external standard curve method can take into account the differences in protein concentration of multiple orders of magnitude, and is suitable for the detection of protein quantitative content in large-scale samples with a large dynamic range.