The SureQuant Quantitation Modules contain 30 unique peptides from 10 AKT-mTOR signaling pathway target proteins for multiplexed quantitation of unique peptides using LC and MS. SureQuant kits utilize multiplex immunoprecipitation coupled with mass spectrometry for simultaneous enrichment and quantitation of target proteins in the AKT/mTOR signaling pathway. Each multiplex panel contains two modules: 1) the IP and MS Sample Preparation Module, which includes all the reagents necessary to immunoenrich target proteins with subsequent in-solution MS sample preparation, and 2) an Absolute or Relative Quantitation Module, including a system suitability standard, and HeavyPeptide™ and LightPeptide™ AQUA Ultimate mixtures.

For relative quantitation, a HeavyPeptide AQUA Ultimate mixture is provided for use as a spike-in internal standard in the multiplexed sample. For absolute quantitation, HeavyPeptide and LightPeptide AQUA Ultimate mixtures are provided with the Pierce™ 6 Protein Digest are used to generate a standard curve in a matrix. The Pierce™ LC-MS/MS System Suitability Standard (7 x 5 mix) is included in both modules to assess the dynamic range of LC-MS/MS systems before running the calibration curves and samples.

The IP and MS Sample Preparation Module enables highly effective antigen IP and co-IP for MS analysis. Cells are first lysed and incubated with an IP-MS–verified biotinylated antibody mixture. The antibody–antigen complex is then captured on MS-compatible streptavidin magnetic beads, and nonspecific proteins are removed through washing with MS-compatible buffers. The target proteins are eluted using trypsin and are compatible with MS sample preparation. The MS sample preparation has been streamlined to be completed in 4 hours so that the sample can be analyzed by MS on the same day.

The AKT/mTOR signaling pathway serves as a master regulatory pathway for cell growth and proliferation and plays a vital role in the signaling cascade. Aberrant activation of this pathway promotes survival of tumor cells in many types of cancer. SureQuant kits are ideal for monitoring AKT/mTOR expression levels, isoforms, and phosphorylation status in a single assay. The ability to simultaneously monitor and quantitate in a single sample is a powerful tool for assessment of normal and abnormal cell growth.

SureQuant AKT Pathway Multiplex Panels are:
• Complete—includes all reagents for successful monitoring of MS system performance, sample preparation, and quantitation of target proteins and peptides
• Verified—antibodies, peptides, and control lysate are rigorously tested for specificity and successful quantitation of each target peptide
• Multiplex—able to quantitate different targets from the AKT/mTOR pathway, RAS, and p53 isoforms
• Flexible—modular format allows for immune-enrichment only, or in combination with relative or absolute quantitation