Why should I include a stage tip step in my PTMScan® experiment?

For PTMScan experiments, the stage tip, or small-scale peptide desalting is a critical part of the experiment for a number of reasons. Detailed instructions for performing stage tip peptide desalting are included at the end of each PTMScan kit protocol.

The stage tip step will remove salts and other contaminants prior to mass spectrometry that can distract the mass spectrometer from peptide identification. Importantly, this step will also serve to reduce the amount of antibody that may co-elute with the peptides and contaminate the analytical column or LC. This will improve the longevity of the analytical column and lead to more reproducible data.

Stage tips can be made using an 18 gauge needle and C-18 disks, according to the method published by Rappsilber et al (Anal. Chem. 2003, 75, 3, 663–670, 2002). Alternatively, the tips can be purchased from a number of suppliers.

The key to a successful desalting step is using a high-capacity stage tip, or one that is suitable for ~10ug of peptide. We find that two C-18 disks or “cookies” packed into the tip are sufficient. It is also important to use freshly made reagents, no more than 2 weeks old, that should be stored in the dark. The Rappsilber method is a spin protocol employing a centrifuge where the reagents and sample are top loaded and spin down through the membrane at the bottom of the tip. For the best results, it is also critical not to carry over any of the agarose/antibody beads from the elution step, as these beads are the major source of background or non-PTM peptides.

Please note that for the elution step we recommend using 40% ACN/0.1% TFA. This optimizes peptide release and minimizes antibody elution from the stage tip. 

Last updated: September 12, 2024