SureQuant Targeted Mass Spec Assay Kits

The Thermo Scientific SureQuant Assays have been designed for multiplexed targeted quantitation for key cellular signaling proteins and include reagents for multiplexed immunoprecipitation and MS sample preparation, heavy peptide quantitation panels, and suitability standards to assess system performance. Together, the SureQuant assay system provides flexibility to use the immunoprecipitation sample prep kits and peptide quantitation modules together, or independently, based on the end-application for robust quantitation.

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We provide complete kits and stand-alone reagents for MS sample preparation and multiplexed enrichment of key signaling pathway proteins. Sample prep for SureQuant analysis can be performed with or without enrichment:

• IP-MS Sample Prep: SureQuant IP-MS Sample Preparation kit (Protein A/G or streptavidin) provides a complete reagent solution for the immunoprecipitation (IP) and mass spectrometry (MS) sample preparation upstream of LC-MS/MS analysis. Cells are lysed in IP-MS cell lysis buffer and incubated with pathway-specific antibody or mixture of user-provided antibodies. The antibody-antigen complex is then captured on protein A/G or streptavidin magnetic beads and the target proteins are eluted and digested with trypsin (Figure 1A). The IP-MS sample preparation module is also available for AKT (total) and AKT (Phospho) signaling pathway proteins.
• General MS Sample Prep: The EasyPep Mini MS Sample Prep Kit enables efficient processing of cultured mammalian cells, tissues and plasma for proteomic mass spectrometry (MS) analysis. The kit is optimized to generate high yield MS-compatible peptide samples in less than 4 hours for direct LC-MS analysis.

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The SureQuant workflow consists of the following steps:

• System Performance: The Pierce LC-MS/MS System Suitability Standard is used to assess MS instrument dynamic range and sensitivity (LLOQ) prior to running calibration curves or unknown samples.
• Sample Prep: The SureQuant sample prep can be done with or without enrichment explained above. The enriched (Figure 1) or unenriched samples (Figure 2) are spiked with internal standards for MS analysis using the SureQuant method.
• MS Data analysis: The Discovery MS data is analyzed with Proteome Discoverer software to assess percent sequence coverage, unique peptides, peptide areas/intensities, and PTMs. For targeted PRM or SureQuant data analysis, Skyline software is used to measure light/heavy ratios and calculate concentrations from unknown samples.

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The AKT/mTOR and RAS/MAPK signaling pathways represent key mechanisms involved in the regulation of cell survival, proliferation, and motility [1] (Figure 2). The cross-talk between two pathways plays a central role in tumor progression and anti-cancer drug resistance [2] (Figure 2). The quantitation of pathway protein expression and modifications are critical for characterization of disease, monitoring cancer progression, and determining treatment response.

Figure 2. Crosstalk between AKT/mTOR and RAS/ERK Pathways.

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Protein kinase B (PKB or AKT) refers to a set of three serine/threonine-specific protein kinases that play a crucial role in cell metabolism, growth, proliferation, and survival. The activation of AKT is controlled by a multi-step process that involves phosphoinositide-3-kinase (PI3K) [3].

• The SureQuant AKT total and phospho pathway panels offer reagents to monitor protein expression of 10 key total and phosphorylated proteins in the AKT signaling pathway (AKT, PTEN, IRS1, IGF1-R, GSK3a, GSK3b, PRAS40, mTOR, p70S6K, TSC2), RAS or TP53 proteins in a single assay (Figure 3 and Figure 4). The SureQuant AKT Isoform module is ideal for monitoring AKT expression levels of the three isoforms (AKT1, AKT2, and AKT3) and phosphorylation status in a single assay (Figure 5).
• The SureQuant panels provide complete quantitation solution for the analysis of key pathway proteins using MS. The Relative or Absolute quantitation modules are available individually or together with IP-MS sample prep kit (multiplex panel).
• The Relative Quantitation Module contains validated AQUA Ultimate HeavyPeptide mixture and serves as a spike-in internal standard for relative quantitation in the multiplexed sample. The Absolute Quantitation Module contains validated AQUA Ultimate HeavyPeptide and LightPeptide mixtures and can be used to determine the absolute concentration of target peptides from AKT-mTOR signaling pathway proteins using targeted MS analysis.

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RAS (GTPase protein) is a key member of the MAPK and PI3K signaling pathways involved in cell growth and proliferation [4].

RAS isoforms (HRAS, NRAS, and KRAS) are mutated in 30% of human cancers The Tumor suppressor protein, TP53 acts as a negative checkpoint regulator for abnormal cell signaling [5]. Many genetic mutations in cancer cells can alter protein expression of RAS and TP53 pathway components.

The SureQuant RAS or TP53 Relative quantitation module enables quantitation of RAS isoforms (HRAS, NRAS, and KRAS) or native and phosphorylated TP53 protein, in a single assay for assessment of normal vs. abnormal pathway signaling (Figure 6A and 6B). For IP-MS sample prep of RAS/TP53, please refer to the section Sample prep for SureQuant analysis.

Figure 6. Relative quantitation of RAS isoforms/TP53 in mammalian cells. HCT116 cells (human colon cancer cell line) treated with IGF1 and untreated BT549 (human breast cancer cell line) were used to measure the relative quantitation of RAS isoforms (A.) and TP53 (B.) using the SureQuant RAS Isoform Panel (Relative Quantitation). The RAS isoforms and TP53 were enriched through multiplex immunoprecipitation followed by nanoLC-PRM/MS analyses on a Q Exactive HF-Orbitrap mass spectrometer.

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• The SureQuant™ Phosphopeptide Suitability Standard contains a mixture of 20 isotopically labeled phosphopeptides with increasing hydrophobic properties for LC optimization and monitoring of LC-MS/MS system performance. The 20 phosphopeptides have a wide range of retention times and the mixture has been designed for use with phosphopeptide samples to assess system performance and chromatography for maximal phosphopeptide identifications (Figure 7).

The SureQuant Multipathway Phosphopeptide Standard contains an optimized mixture of 131 isotopically labeled phosphopeptides selected to cover biologically relevant phosphorylation sites for 89 key proteins from seven signaling pathways including EGFR/HER, RAS-MAPK, PI3K/AKT/mTOR, AMPK, death/apoptosis, and stress (p38/SAPK/JNK). The workflow for sample analysis using SureQuant Multipathway standard is shown in Figure 8(A). The use of Multipathway Standard with phosphopeptide enrichment enables simultaneous monitoring and quantitation of target proteins in a single sample for assessment of normal and abnormal signaling pathway activity (Figure 8(B)).

Figure 8. Multipathway Phosphopeptide Standard Method Optimization. A. Workflow. B. Heat map of relative levels of phosphopeptides compared between treated cell lysates (left). Changes in specific phosphopeptides (right). One μg of treated cell lines was incubated with High-Select Fe-NTA magnetic beads. Prior to enrichment, 1 pmol of the SureQuant Multipathway Phosphopeptide Standard was spiked into the lysate. PRM analysis was performed using the Dionex UltiMate 3000 RSLCnano System and Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer, with subsequent analysis in Skyline software using the average total area. A: HCT116/IGF; B: MCF7/IGF;
C: LNCAP/IGF; D: A431/EGF; E: HepG2/Ins.

• The Pierce LC-MS/MS System Suitability Standard (7 x 5 mix) contains five sets of seven HeavyPeptide AQUA Ultimate peptides distinguished by unique isotopologue labeling to assess system performance of LC-MS/MS systems (Figure 9(A)). The seven peptides are common with 15 peptides of Pierce Peptide Retention Time Calibration Mixture. Each of the seven peptides has five versions (amino acids labeled with 0, 1, 2, 3, or 4 heavy isotopes), at five distinct concentrations (Figure 9(B)) and can be used with Skyline software to assess dynamic range, linearity, and lower limit of quantitation (LLOQ).

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Explore our webinars around targeted mass spectrometry. Click on targeted proteomics section to find these webinars that discuss targeted mass spectrometry workflows and its applications to quantitate proteins of interest. Various strategies to reduce sample complexity through improved sample preparation and enrichment, providing guidance towards targeted MS-assay development, will be introduced.

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1. Mendoza M.C., Er E.E., Blenis J. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem Sci, 2011.
   36(6):320–328. 
2. Al-Olabi L., Polubothu S., Dowsett K., et al. Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy. J Clin Invest, 2018. 128(4):1496–1508. 
3. Hemmings B.A., Restuccia D.F. PI3K-PKB/Akt pathway. Cold Spring Harb Perspect Biol, 2012. 4(9):a011189. 
4. Krygowska A.A., Castellano E. PI3K: A Crucial Piece in the RAS Signaling Puzzle. Cold Spring Harb Perspect Med, 2018. 8(6):a031450. 
5. Kastenhuber ER, Lowe SW. Putting p53 in Context. Cell, 2017. 170(6):1062–1078. 

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