The PTMScan^® Control Peptides Phospho-Akt (RXXS*/T*) enable quality control of immunoaffinity enrichment performance using PTMScan^® or PTMScan^® HS workflows.  These synthetic peptides contain a specific post-translational modification (PTM) that can be enriched by the associated PTMScan^® or PTMScan^® HS immunoaffinity purification (IAP) beads, as well as a stable heavy isotope that can be distinguished from endogenous peptides by the mass spectrometer. 

Akt plays a central role in mediating critical cellular responses, including cell growth and survival, angiogenesis, and transcriptional regulation (1-3). It is a member of an important class of kinases, referred to as Arg-directed kinases or AGC-family kinases, which includes cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C, Akt, p70 S6 kinase, and RSK. These kinases share a substrate specificity characterized by Arg at position -3 relative to the phosphorylated Ser or Thr (4,5). Akt, p70 S6 kinase, and RSK additionally share specificity for Arg at position -5 (recognition sequence RXRXXS/T) (6). In a recent phosphoproteomic study (7) co-authored by scientists in the Cell Signaling Technology Site Discovery Group, over 300 downstream substrates for AGC-family kinases recognizing the RXRXXS/T motif were identified with PTMScan^® Technology using Phospho-Akt substrate antibodies. These Cell Signaling Technology antibodies are powerful tools for investigating the regulation of phosphorylation by Akt and other Arg-directed kinases, as well as for high throughput kinase drug discovery.

In this assay, PTMScan^® (RXXS*/T*) Motif Antibody bead conjugates are used to specifically enrich phosphopeptides containing the RXXS*/T* motif (S* = phospho-serine, T* = phospho-threonine).

Use with Cell Signaling Technology’s PTMScan^® kit protocol from the Immunoaffinity Purification (IAP) step.  Because the optimal amount of PTMScan^® Control Peptides Phospho-Akt (RXXS*/T*) for each user’s experiments will depend on unique factors, such as mass spectrometer sensitivity, users may dilute these control peptides as needed.

1. Aliquot PTMScan^® Control Peptides Phospho-Akt (RXXS*/T*) for storage as single-use units at -20°C or proceed to immediate usage.  
2. Resuspend sample peptides in the appropriate buffer and volume, e.g., 1.4 mL of PTMScan^® IAP Buffer (1X).
3. Clear sample peptides by centrifugation.
4. Transfer clarified sample peptides to tubes containing IAP beads.
5. Add 10 µL of PTMScan^® Control Peptides Phospho-Akt (RXXS*/T*) to IAP beads and sample peptides and mix well. 
6. Continue with PTMScan^® or PTMScan^® HS workflows at the 2-hour incubation step.
7. Detect PTMScan^® Control Peptides Phospho-Akt (RXXS*/T*) in the LCMS data file.