Default Home     Favorite

Protein Quantification Analysis

    Label-Free Quantification

This method is especially suited for biomarker discovery in large sample sets because it does not cost so much like labeled quantification and has an accurate quantification result similar with labeled quantification. We have developed a fully automated label-free approach to quantify proteins from LC-MS/MS experiments. Based on this, we offer label-free proteomics services using High Definity Mass Spectrometry under software PLGS 2.3. We have successfully performed a large number of LC-MS/MS quantification projects with excellent chromatography reproducibility for our customers.

    Isotope labeled Quantification

 o Multiplexed Isobaric Tagging Technology (iTRAQ)

iTRAQ Technology (Multiplexed Isobaric Tagging Technology) uses a chemical tagging reagent which allows multiplexing of two to eight protein samples and produces identical MS/MS sequencing ions for all eight versions of the same derivatized tryptic peptide. This greatly facilitates peptide identification due to resulting higher intensities of the parent and fragment ions. Quantitation is achieved by comparison of the peak areas and resultant peak ratios for either four MS/MS reporter ions, which range from 114 to 117 Da or eight MS/MS reporter ions, which range from 113-119 and 121 Da. The relative areas of the reporter ion peaks provide the expression ratios of the parent proteins.

                                     Mol Cell Proteomics. 2004;3(12):1154-1169.

 o Isotope Coded Affinity Tag Technology (ICAT)

The ICAT reagent reacts with free cysteines in proteins in cell lysates and contains an affinity tag (biotin) and an acid cleavage site (for biotin removal). The tag is isotopically coded for a “heavy” or “light” tag, with 9 amu mass difference. After reacting 100ug control protein extract with the “light” tag and an equal amount of experimental protein extract with the “heavy” tag; the samples are mixed, trypsin digested, and cysteine-containing peptides are affinity purified on an avidin column. While ICAT has the advantage of simplifying complex extracts, it cannot detect proteins that lack cysteine. The purified, cysteine containing tryptic peptides are cleaved with trifluoroacetic acid to remove the biotin group and parent proteins are identified using LC-MS/MS. Quantitation is carried out with relevant software with the results.

                   Nat. Biotechonl. 1999,17:994-998