Why are there background peptides in my PTMScan® experiment?

The background or number of non-PTM peptides in each enrichment is primarily due to non-specific interactions with the beads the antibody is conjugated to. Therefore, this background is also non-depletable by methods such as pre-clearing.

The level of background peptides can range considerably depending on the PTM and the biological source. PTMs that are typically lower in abundance (e.g., phosphotyrosine) can be expected to have a higher background compared to more abundant PTMs like the ubiquitin remnant (or K-e-GG) and acetyl lysine. Peptides derived from cells in culture typically produce more PTM peptides than an equivalent amount of protein from tissue.

A major source of background peptides can be carry-over of antibody-beads to the stage tip or small-scale desalting at the end of the protocol. For optimal results, the eluted peptides should be clear of any antibody-beads before loading onto the stage tip. The pH of the peptides in the IAP enrichment step may also affect the specificity. The antibodies are pH sensitive and for optimal results, each peptide suspension should be a pH of ~7.

Last updated: February 27, 2024