Mass spectrometry techniques can be used to profile post-translationally modified (PTM) peptides in a digested sample. However, PTM peptides are often present at very low abundance, which is a challenge when trying to study them using mass spectrometry techniques.
To address this challenge Cell Signaling Technology (CST™) has developed PTMScan® technology, a proprietary proteomic method that combines antibody enrichment of PTM-containing peptides with liquid chromatography mass spectrometry (LC-MS/MS). PTMScan technology allows identification and quantification of hundreds to thousands of even the lowest abundance peptides, and provides a more focused approach to peptide enrichment than other strategies (for example IMAC).
PTMScan technology can be used to:
The antibodies used to enrich PTM-containing peptides are key to the success of PTMScan® technology. They are:
The table below outlines the three types of antibodies employed in PTMScan technology.
|Standard site-specific PTM antibody||Modified amino acid in the context of a specific sequence of amino acids surrounding it.||A CST™ antibody to Akt1 phosphorylated at serine 473 only recognizes that particular phosphoserine and the surrounding amino acids.|
|Motif antibody||Modified amino acid within a certain motif.||The Akt substrate motif antibody will recognize the sequence RXRXXS* in any protein only when the serine residue is phosphorylated (where X can be any amino acid).|
|PTM-specific antibody (PTM-antibody)||Any peptide with the PTM of interest.||A CST acetyl-lysine antibody will recognize all acetylation sites independent of flanking amino acid sequences.|
|PTMScan® Discovery |
|PTMScan® Direct |
(mass spectrometry-based antibody array)
|Is a specific pathway targeted?||✗||✔|
|Is antibody enrichment performed?||✔||✔|
|Is LC-MS/MS performed?||✔||✔|
|What type of antibodies are used?||PTM or motif antibodies to undefined targets.||Standard site-specific antibodies to defined targets within the known pathway(s) of interest.|
|What is the bead format?||Antibodies against one PTM or motif on each bead.||Antibodies against many targets on each bead (a bead-based multiplex assay).|
|Which species can you use?||Can be used on samples from many different species including, but not limited to, human, mouse, rat, drosophila, and arabidopsis.||Validated for human and mouse. (Contact us for other species.)|
|Case Study||“Deep, quantitative coverage of the acetylome using novel anti-acetyl-lysine antibodies and an optimized proteomic workflow.” |
Svinkina, T., et al (2015) Mol. Cell. Proteomics 14(9):2429–40.
|“PTMScan Direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS.” |
Stokes, M., et al (2012) Mol Cell Proteomics. 11(5):187–201.
|Summary||Use PTMScan Discovery to find new information with quantitative analysis of PTMs.||Use PTMScan Direct to quantitatively assay the activity of components of known signaling pathways across cell lines or treatments.|