A need exists for technologies that allow direct quantification of differences in protein expression levels. There is a strategy for absolute quantification (termed AQUA) of proteins and their modification states. AQUA, enabling absolute protein quantitation using stable isotope-labeled peptides and HPLC-MS. By applying a common principle, a labeled molecule as an internal standard, to protein analysis, AQUA has enhanced the abilities of protein researchers to study complex biological samples quantitatively and provided a valuable new tool for proteomics. For proteomics researchers, it facilitates targeted, quantitative studies of not only specific protein expression, but specific amino acid modification as well.
Our AQUA strategy has two stages: peptide internal standard selection and validation, and method development and implementation.
In the first stage, a representative tryptic peptide is selected and standard synthesized from the protein of interest with an amino acid sequence that exactly mimics the corresponding native peptide produced during proteolysis. And stable isotopes are incorporated during the synthesis process. Then, the synthetic AQUA internal standard peptide is next evaluated by LC-MS/MS. The SRM analysis is then performed to measure a specific precursor-to-product ion transition.
Figure 1. AQUA peptide development
The second stage of the AQUA strategy is its implementation to measure the amount of proteins or modified proteins in a complex mixture. Cell lysates can be optionally separated prior to proteolysis to increase the dynamic range of the assay. We have most commonly used SDS-PAGE for this enrichment, followed by excision of the region of the gel where the protein of interest migrates. In-gel digestion is performed in the presence of the AQUA peptide, which is added to the gel pieces during the digestion process. Following proteolysis, the complex peptide mixture, containing both heavy and light peptides, is analyzed in an LC-SRM experiment using parameters determined during Stage 1.
Figure 2. Method implementation.
Creative Proteomics also provide the following bioinformatics services in Absolute Quantification:
Our team is ready to support you and works with you to develop a research plan for absolute quantification. Our projects are carried out in our state of the art proteomics facility. Please feel free to contact us to discuss your projects. We hope you will find that we can meet your research needs.