Absolute Quantification (AQUA) Service

What is AQUA?

The AQUA technique is a widely employed method for absolute quantification of proteins and peptides, which relies on the isotope-labeled internal standard peptide (ILISP) and Liquid Chromatography-Mass Spectrometry (LC-MS). It was first proposed and utilized by Gerber et al. in 2003. The common isotopic elements include 18O, 13C, 2H or 15N, etc. The AQUA methods mainly including selection of suited ILISP for target proteins and the development of optimized LC-MS/MS methods. Chemically synthesized ILISP which mimic the native peptides of interest generated through proteolysis are added into a complex sample (e.g., cell lysates) in known amounts during digestion. The native peptide and ILISP possess identical amino acid sequences, thereby sharing physicochemical properties such as chromatographic retention time, ionization efficiency, fragmentation mechanism, and relative distribution of fragment ions. However, differentiation between the native and ILISP peptides can be achieved through the introduced mass shift (Approximate 4-10 Da in the mass.). Consequently, the mixture proteolyzed of native peptides and ILISP enable simultaneous LC-MS analysis and peak area detection of both peptides. As the known amount of the ILISP, the amount of native target protein can be subsequently calculated by comparing the respective peak areas. Moreover, the synthetic peptides can also be modified through covalent modifications, such as phosphorylation, ubiquitination, methylation, acetylation, etc., which are chemically indistinguishable from naturally occurring post-translational modifications (PTMs). Therefore, MS-based AQUA strategy is developed so powerful that it can accurately precise quantify both the concentration of protein expression and their PTMs levels.

Importantly, the AQUA methodologies enable the precise determination of protein concentrations in biological specimens, including cells, tissues, and bodily fluids. Due to its ability to provide precise and accurate absolute quantification, it is essential for specific applications such as the evaluation of clinical biomarker candidates and the comprehension of protein biological function.

The widely applications of the AQUA approach in proteomics ^[1].

1. Evaluation of protein expression, degradation rates, and variability, and definition of PTMs levels in model biological systems;
2. Study of cellular organization and dynamics;
3. Determination of protein and their PTMs stoichiometries and ratio variability within protein complexes;
4. Study of changes of protein levels and activation status in protein networks;
5. Validation of potential new biomarkers and their eventual clinical utility;
6. Study of potential protein therapeutic agents’ stability;
7. Investigation of enzymes kinetics;
8. Development of optimum drug delivery;
9. Pharmacokinetic studies on the behaviors of compounds during drug development.
10. Antibiotic stress research for the development of new drugs.
11. Quantification and differentiation of highly homologous protein isoenzymes research.
12. Low abundance protein.
13. Food safety inspection for food allergic protein (Allergens).

The selection principle of the ILISP for the proteins of interest.

1. The length of peptide is less than 15 amino acid.
2. The sequence must be unique within the entire proteome.
3. The sequence does not contain sites prone to biochemical modification such as methionine and cysteine;
4. It does not contain special sequence forms such as the aspartic acid-glycine sequences that are easily broken by enzymatic peptide bonds and deamidated N-terminal glutamine.

Table 1. The currently used heavy amino acids in synthetic peptides.

The basic workflow of the AQUA (MRM/MS) method for absolute protein quantification.

1. Selection of proper peptide fragments for proteins of interest;
2. The ILISP synthesis;
3. Confirm the ILISP - Whether the chromatographic behavior and fragmentation spectra align with the previously detected or predicted characteristics;
4. Choice of MS instrument and analysis method;
5. Method optimization;
6. Sample preparation - Known amounts of protein lysates and ILISP are mixed together and subjected to protease digestion.
7. MS analysis - Analysis of the proteolyzed sample by a Multiple reaction monitoring (MRM) experiment in a LC-MS/MS technique.

Figure 1. General scheme of the AQUA strategy ^[2].

The Advantages on Creative Proteomics to implement AQUA-SRM Service.

1. Highly accurate and reliable quantification results: The utilization of advanced AQUA technology, which incorporates the ILISP, ensures the attainment of precise and reproducible results (variation CV<5%).
2. High applicability of various sample types: Such as cells, tissues, body fluids, etc.
3. Professional detection and analysis capability: Experienced MRM target quantification proteomics team, strict quality control system, together with ultra-high resolution detection system and professional data pre-processing and analysis capability, ensure reliable and accurate data.
4. Reproducible: Obtain consistent and reproducible inter- and intra- assay results for data analysis.
5. High throughput and sensitivity: Q Exactive, Q Exactive HF, Orbitrap Fusion™ Tribrid™, QTrap6500, etc.
6. The results are delivered in a short term and of high quality.

How to place an order

With over 10 years experimental experience, Creative Proteomics have the expertise to provide a tailored and scientific experimental scheme that meets your specific requirements for conducting the Absolute Quantification (AQUA) service, facilitating precise and accurate identification and quantification of target proteins or peptides. Please feel free to contact us to discuss your specific needs. Our customer service representatives are available 24 hours a day, from Monday to Sunday.

References

1. Calderón-Celis F, Encinar JR, Sanz-Medel A. Standardization approaches in absolute quantitative proteomics with mass spectrometry. Mass Spectrometry Reviews. 2018 Nov;37(6):715-737.
2. Küchler J, Püttker S, Lahmann P, et al. Absolute quantification of viral proteins during single-round replication of MDCK suspension cells. Journal of Proteomics. 2022 May 15;259:104544.
3. Kirkpatrick DS, Gerber SA, Gygi SP. The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods. 2005 Mar;35(3):265-73.

Analysis and Interpretation of Protein Post-Translational Modification Site Stoichiometry

Journal: Trends in Biochemical Sciences

Published: 2019

Main Technology: absolute quantification (AQUA), Label-Free Quantification, stable isotope labeling by amino acids in cell culture (SILAC)-based approach etc.

Abstract

Proteins are decorated with a diverse array of post-translational modifications (PTMs) that regulate their spatial and temporal functions. Recent mass spectrometry (MS)-based studies have identified hundreds of thousands of PTM sites in mammalian proteomes. However, the signaling cues and enzymes regulating individual sites are often not known and their functional roles remain uncharacterized. Quantification of PTM site stoichiometry can help in prioritizing sites for functional analyses and is important for constructing mechanistic models of PTM-dependent protein regulation. Here, we review the concept of PTM site stoichiometry, critically evaluate the merits and drawbacks of different MS-based methods used for quantifying PTM site stoichiometry, and discuss the usefulness and limitations of stoichiometry in informing on the biological function of modified sites.

Figure 1. Analysis of PTM Site Stoichiometry by AQUA and Label-Free Quantification.
(A) AQUA-based quantification. (B) Label-free strategy.

For figure 1(A), a known concentration of synthetic, stable isotope-labeled modified and unmodified peptides (red) is spiked into a sample containing native peptides. The MS intensity of the AQUA peptides is compared with the intensity of native modified and unmodified peptides; due to isotope-labeling, the mass to charge (m/z) ratio differs between AQUA and native peptides. By knowing the absolute concentration of AQUA peptides, the abundance of native modified peptides and unmodified peptide can be quantified and PTM stoichiometry can be calculated using the indicated formula.

1. The drawback of AQUA.

a. Stringent sequence selection criteria reduce the range of available peptides;

b. AQUA isotope-labeled internal standard peptides (ILISPs) are provided in a lyophilized state, and variations in solubility may result in unstable experimental results;

c. AQUA ILISPs need to be synthesized, purified and quantified respectively, which incurs significant costs for large-scale protein quantification.

2. The distinction between AQUA and alternative methods for stable isotope labeling protein quantification^[3].

3. The Known concentrations of ILISPs are added to the cell lysates before or after protease digestion.

It is generally recommended to incorporate ILISPs into cell lysate prior to protease digestion, as indicated by most reports. Because it could mimic corresponding proteins in a reality state that facilitates accurate quantification.