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Proteomics

Proteomics

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:

  • Determine novel protein sites that are phosphorylated, ubiquitinated, acylated, methylated, or protease cleaved.
  • Identify and validate drug targets.
  • Discover biomarkers.
  • Explore the mechanism of action of drugs/chemical modulators.
  • Elucidate off-target drug effects.
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Antibodies for PTMScan

The antibodies used to enrich PTM-containing peptides are key to the success of PTMScan® technology. They are:

  • Designed and produced in-house
  • Rigorously tested for specificity, sensitivity, and consistency
  • Specially formulated for optimal immunoaffinity enrichment

The table below outlines the three types of antibodies employed in PTMScan technology.

Antibody type Recognizes Example  
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. Standard site-specific PTM antibody Sequence
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). Motif Antibody Sequence
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. PTM-specific antibody (PTM-antibody) Sequence

Which option is right for your research?

  PTMScan® Discovery
(PTM/motif-based enrichment)		 PTMScan® Direct
(mass spectrometry-based antibody array)
  PTMScan Discovery PTMScan Direct
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.

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