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.
Antibody type | Recognizes | Example | |
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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 (PTM/motif-based enrichment) | PTMScan® Direct (mass spectrometry-based antibody array) | |
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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. |