Proteomics, the large-scale analysis of proteins, has become an increasingly important tool for identifying and characterizing novel drug targets, as it provides a powerful way to understand disease mechanisms, discover new biomarkers, and profile drug pharmacology. PTMScan® is an immunoaffinity-based method developed by CST scientists for identifying and quantifying peptides with post-translational modifications (PTMs) that employs PTM-specific antibodies and liquid chromatography with tandem mass spectrometry (LC-MS/MS).
Further Reading: A Revolution in Proteomics Analysis: PTMScan 20 Years Later
As with any mass spectrometry experiment, maintaining consistency between proteomic experiments is of critical importance in order to ensure comparable results. From consistency in sample handling, to affinity performance, to instrument sensitivity, there are a variety of factors that can affect results, especially when experiments are being performed weeks or months apart. Ensuring reproducibility improves your ability to statistically evaluate changes in the abundance of PTM sites across conditions, without having to resort to unreasonably large sample sizes and replicates.
To help scientists more easily ensure reproducibility in their proteomics experiments, we have created a class of peptide standards that can be used to monitor the efficiency and consistency of the PTMScan enrichment: PTMScan Control Peptides. These standards are synthetic peptides that are based on naturally generated tryptic sequences. The key feature is the use of “heavy” lysine (13C6 15N2-Lysine) or arginine (13C6 15N4-Arginine) amino acids during peptide synthesis. These heavy versions of the peptides are easy to discriminate from the endogenous, light form because of their inherent mass difference due to the incorporation of the heavy lysine and arginine amino acids. This can be seen in Figure 1, which shows the extracted ion chromatogram of heavy vs light peptides for the ubiquitin remnant antibody control peptides. It is important to note that not every cell or tissue type will endogenously express a given modified peptide sequence, but such a sample is still amenable for spike-in of heavy control peptides.Figure 1. The extracted ion chromatograms (EIC) of the PTMScan Control Peptides Ubiquitin/SUMO #75964 (blue) spiked into a tryptic digest of mouse liver following enrichment with the PTMScan HS Ubiquitin/SUMO Remnant Motif Kit #59322. The heavy control peptides are readily discernible from the co-eluting endogenous light-modified peptides (black) present in the mouse liver sample due to the mass difference of the heavy isotopes used. The identification of endogenous modified peptides (Figure 1B) in the mouse liver sample using the PTMScan kit is not affected by the addition of the PTMScan Control Peptides.
How to Use Control Peptides in PTMScan Discovery Experiments
Each peptide control mix contains at least three heavy peptides that are specific to the corresponding PTMScan kit and that have been tested extensively in control material to ensure they are consistently enriched by the PTMScan reagents under a variety of conditions. The control peptides in each mixture were chosen based on their elution time so that they span the chromatographic gradient, as well as their ionization properties to ensure sufficient signal-to-noise ratio.
To use the control mix, an aliquot of the control peptide mixture is spiked into the experimental sample prior to performing the corresponding PTMScan enrichment. Each control peptide mix comes with enough peptides for 10 PTMScan experiments.
As shown in Figure 1, following affinity enrichment and mass spectrometry, each control peptide can be distinguished from its potentially present endogenous form by extracted ion chromatography (EIC) due to its unique mass/charge ratio.
Each kit specifies the calculated, singly protonated, precursor mass (MH+) of the control mix and the corresponding recommended mass-to-charge measurement (m/z) for each heavy peptide in the control peptide mixture to streamline identification and quantification. Using this information, you can easily compare the observed and calculated precursor measurement (m/z) of the control peptides to assess the mass accuracy of your instrument, allowing you to adjust the mass accuracy tolerance for your database search and PTM peptide identifications. The annotated tandem mass spectra (MS/MS) of each control peptide component are also specified for each mix, as shown in Figure 2. This can be used to compare the observed MS/MS with the provided MS/MS to assess whether fragmentation parameters provide sufficient ion sensitivity and coverage to identify and localize PTM sites.
Figure 2. Annotated tandem mass spectrum (MS2) of the PTMScan Control Peptides Ubiquitin/SUMO #75964 component: TITLEVEPSDTIENVK(gg)A[K].
Our extensive testing has shown that the addition of these control peptides at the recommended levels does not negatively affect the enrichment efficiency or recovery of endogenous PTM peptides. Using area under the curve (AUC) measurements, the abundance of each peptide can be quantified and compared across multiple experiments, providing critical information about the efficiency and reproducibility of the enrichments. Furthermore, because the PTMScan reagents are highly specific, experiments employing sequential enrichment strategies from one sample are possible, and the control peptides can be used at each individual enrichment step to monitor results.
Figure 3. Recovery of KGG Control Peptides across three independent PTMScan HS Ubiquitin experiments using 1mg of human HEK293 sample peptides. Data were analyzed on ThermoFisher Fusion Lumos.
It’s important to note that the control peptide mixture can be used irrespective of the species under investigation. Using an accurate mass instrument, the unique mass/charge ratio in combination with elution time allows the researcher to identify these peptides in any PTMScan experiment. When using the control peptides in samples of other species, it is important to be aware that there may not be an endogenous signal due to sequence variation of the protein in the other species.
Overall, these control peptides serve as an external control that can be used in real-time to help assess reproducibility with the affinity enrichment and peptide desalting steps for a wide range of PTMScan experiments.
PTMScan Control Peptides
CST offers control peptides for many of the PTMScan enrichment kits available, and we highly recommend using the associated control peptide in your next PTMScan experiment. See the table below for a list of products, each of which is compatible with PTM enrichment workflows, including both agarose and magnetic bead-based formats.