Kit containing SureQuant AKT Pathway IP and MS Sample preparation reagents

Targeted quantitation is performed in proteomic experiments to increase the sensitivity of detection for a limited number of target analytes. These approaches require spiking a sample with known amounts of synthetic peptides containing heavy stable isotopes, which act as internal quantitative standards for quantitation of the corresponding natural peptides in the sample. The main types of targeted quantitation methods include single reaction monitoring (SRM) and parallel reaction monitoring (PRM). SureQuant PRM is the next generation of assays that include a suite of kits and standards to enable multiplexed quantitation of key cellular targets.

See SureQuant selection guide

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.

Selection guide for SureQuant reagents

The AKT pathway IP-MS sample preparation kit is used to immuno-enrich total target proteins of AKT-mTOR pathway with subsequent in-solution MS sample preparation. The quantitation module enables relative quantitation of unique peptides of AKT pathway proteins using LC-MS.

Learn more about AKT pathway (total) reagents

Workflow stepProductTarget proteinsUnit sizeCat. No.
Sample prepSureQuant AKT Pathway IP and MS Sample Preparation Module

AKT/mTOR pathway:


AKT1, AKT2, AKT3, PTEN, IRS1, IGF1-R, GSK3a, GSK3b, PRAS40, mTOR, p70S6K, TSC2; RAS and p53 isoforms
10 reactionsA40081
Protein quantitationSureQuant AKT Pathway Relative Quantitation Module10 reactionsA40082
Sample prep and Protein quantitationSureQuant AKT Pathway Multiplex Panel (Relative Quantitation)10 reactionsA40080

The AKT pathway (phospho) IP-MS kit is used for multiplexed enrichment and quantitation of key phosphorylated proteins in the AKT-mTOR signaling pathway. The quantitation modules enable either relative or absolute quantitation of unique peptides of AKT pathway phospho proteins using LC-MS.

Learn more about AKT pathway (phospho) reagents

Workflow stepProductPhosphorylated TargetsUnit sizeCat. No.
Sample prepSureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module

AKT/mTOR pathway:


AKT1 pSer473, AKT2 pSer474, PTEN pSer380, IRS1 pSer312, IGF1-R pTyr1162/1163, GSK3a pSer21, GSK3b pSer9, PRAS40 pThr246, mTOR pSer2448, p70S6K pThr389, TSC2 pSer939; RAS and p53 isoforms
10 reactionsA40086
Protein quantitationSureQuant AKT Pathway (Phospho) Relative Quantitation Module10 reactionsA40087
Sample prep and Protein quantitationSureQuant AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation)10 reactionsA40088
Sample prep and Protein quantitationSureQuant AKT Pathway (Phospho) Multiplex Panel (Relative Quantitation)10 reactionsA40084
Sample prep and Protein quantitationSureQuant AKT Pathway (Phospho) Multiplex Panel (Relative Quantitation)10 reactionsA40085

The AKT isoform relative quantitation kit enables comparative quantitation of AKT1, AKT2, AKT3 isoforms and phosphorylation of AKT using LC-MS.

Learn more about AKT pathway (isoform) reagents

ProductTarget proteinsUnit sizeCat. No.
SureQuant AKT Isoform Relative Quantitation ModuleAKT1, AKT2 and AKT3 isoforms10 reactionsA40092

The RAS isoform or TP53 relative quantitation kit is used for relative quantitation of NRAS, KRAS and HRAS isoforms or native and phosphorylated TP53 target proteins using LC-MS. For sample prep, see Standards and Sample prep tab.

Learn more about RAS isoform and TP53 reagents

ProductTarget proteinsUnit sizeCat. No.
SureQuant RAS Isoform Relative Quantitation ModuleNRAS, KRAS and HRAS isoforms10 reactionsA40097
SureQuant TP53 Panel Relative Quantitation ModuleTP53 (native and phosphorylated)10 reactionsA40102

The MS system suitability standards are used for MS instrument calibration to assess system performance and the phosphopeptide standard is utilized to maximize phosphopeptide identifications. The IP-MS sample prep kits can be used for recovery of low-abundant target proteins, and the EasyPep kits can be used for samples not requiring enrichment.

Learn more about MS standards and sample prep

Workflow stepProductProteins/Signaling pathwaysUnit sizeCat. No.
Chromatography optimization and system performance for phosphopeptidesSureQuant Phosphopeptide Suitability StandardSerine, threonine and tyrosine phosphorylated50 µLA51746
Phosphopeptide quantitationSureQuant Multipathway Phosphopeptide StandardPI3K/AKT/mTOR; EGFR/HER; RAS-MAPK; AMPK; Death/Apoptosis; Stress (p38/SAPK/JNK)200 µLA51745

General MS standard and SureQuant sample prep reagents.

Workflow stepProductUnit sizeCat. No.
LC-MS system calibrationPierce LC-MS/MS System Suitability Standard (7 x 5 mixture)25 µLA40010
100 µLA51747
Sample prepSureQuant Protein A/G IP-MS Sample Preparation Kit
20 reactionsA51743
Sample prepSureQuant Streptavidin IP-MS Sample Preparation Kit20 reactionsA51744
Sample PrepEasyPep MS sample prep kit8 reactionsA45734
20 reactionsA40006
96 reactionsA45733

The AKT pathway IP-MS sample preparation kit is used to immuno-enrich total target proteins of AKT-mTOR pathway with subsequent in-solution MS sample preparation. The quantitation module enables relative quantitation of unique peptides of AKT pathway proteins using LC-MS.

Learn more about AKT pathway (total) reagents

Workflow stepProductTarget proteinsUnit sizeCat. No.
Sample prepSureQuant AKT Pathway IP and MS Sample Preparation Module

AKT/mTOR pathway:


AKT1, AKT2, AKT3, PTEN, IRS1, IGF1-R, GSK3a, GSK3b, PRAS40, mTOR, p70S6K, TSC2; RAS and p53 isoforms
10 reactionsA40081
Protein quantitationSureQuant AKT Pathway Relative Quantitation Module10 reactionsA40082
Sample prep and Protein quantitationSureQuant AKT Pathway Multiplex Panel (Relative Quantitation)10 reactionsA40080

The AKT pathway (phospho) IP-MS kit is used for multiplexed enrichment and quantitation of key phosphorylated proteins in the AKT-mTOR signaling pathway. The quantitation modules enable either relative or absolute quantitation of unique peptides of AKT pathway phospho proteins using LC-MS.

Learn more about AKT pathway (phospho) reagents

Workflow stepProductPhosphorylated TargetsUnit sizeCat. No.
Sample prepSureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module

AKT/mTOR pathway:


AKT1 pSer473, AKT2 pSer474, PTEN pSer380, IRS1 pSer312, IGF1-R pTyr1162/1163, GSK3a pSer21, GSK3b pSer9, PRAS40 pThr246, mTOR pSer2448, p70S6K pThr389, TSC2 pSer939; RAS and p53 isoforms
10 reactionsA40086
Protein quantitationSureQuant AKT Pathway (Phospho) Relative Quantitation Module10 reactionsA40087
Sample prep and Protein quantitationSureQuant AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation)10 reactionsA40088
Sample prep and Protein quantitationSureQuant AKT Pathway (Phospho) Multiplex Panel (Relative Quantitation)10 reactionsA40084
Sample prep and Protein quantitationSureQuant AKT Pathway (Phospho) Multiplex Panel (Relative Quantitation)10 reactionsA40085

The AKT isoform relative quantitation kit enables comparative quantitation of AKT1, AKT2, AKT3 isoforms and phosphorylation of AKT using LC-MS.

Learn more about AKT pathway (isoform) reagents

ProductTarget proteinsUnit sizeCat. No.
SureQuant AKT Isoform Relative Quantitation ModuleAKT1, AKT2 and AKT3 isoforms10 reactionsA40092

The RAS isoform or TP53 relative quantitation kit is used for relative quantitation of NRAS, KRAS and HRAS isoforms or native and phosphorylated TP53 target proteins using LC-MS. For sample prep, see Standards and Sample prep tab.

Learn more about RAS isoform and TP53 reagents

ProductTarget proteinsUnit sizeCat. No.
SureQuant RAS Isoform Relative Quantitation ModuleNRAS, KRAS and HRAS isoforms10 reactionsA40097
SureQuant TP53 Panel Relative Quantitation ModuleTP53 (native and phosphorylated)10 reactionsA40102

The MS system suitability standards are used for MS instrument calibration to assess system performance and the phosphopeptide standard is utilized to maximize phosphopeptide identifications. The IP-MS sample prep kits can be used for recovery of low-abundant target proteins, and the EasyPep kits can be used for samples not requiring enrichment.

Learn more about MS standards and sample prep

Workflow stepProductProteins/Signaling pathwaysUnit sizeCat. No.
Chromatography optimization and system performance for phosphopeptidesSureQuant Phosphopeptide Suitability StandardSerine, threonine and tyrosine phosphorylated50 µLA51746
Phosphopeptide quantitationSureQuant Multipathway Phosphopeptide StandardPI3K/AKT/mTOR; EGFR/HER; RAS-MAPK; AMPK; Death/Apoptosis; Stress (p38/SAPK/JNK)200 µLA51745

General MS standard and SureQuant sample prep reagents.

Workflow stepProductUnit sizeCat. No.
LC-MS system calibrationPierce LC-MS/MS System Suitability Standard (7 x 5 mixture)25 µLA40010
100 µLA51747
Sample prepSureQuant Protein A/G IP-MS Sample Preparation Kit
20 reactionsA51743
Sample prepSureQuant Streptavidin IP-MS Sample Preparation Kit20 reactionsA51744
Sample PrepEasyPep MS sample prep kit8 reactionsA45734
20 reactionsA40006
96 reactionsA45733

Sample Prep for SureQuant analysis

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.

SureQuant workflow

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.

 

Schematic representation of SureQuant workflow

Figure 1. Pathway analysis using Surequant workflow. A. IP-MS/SureQuant workflow uses multiplexed IP enrichment followed by MS sample preparation. B. SureQuant sample preparation using EasyPep Maxi for generation of peptide digests for unenriched samples. Both SureQuant workflows enable multiplexed quantitation of target proteins.

AKT/mTOR and RAS/MAPK pathway overview

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.

Schematic representation of AKT/ mTOR and RAS/ERK pathways

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

AKT pathway (total) (phospho) and isoform reagents

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.
Graph showing relative quantitation of AKT/mTOR pathway proteins in A549 cells.

Figure 3. Relative quantitation of AKT/mTOR pathway proteins in in mammalian cells. A549 cells (human adenocarcinoma cell line) were treated with IGF1 and the relative quantitation of AKT/mTOR pathway proteins was determined using the SureQuant AKT Pathway Multiplex Panel (Relative Quantitation). The pathway proteins were enriched through multiplex immunoprecipitation followed by nanoLC-PRM/MS analyses on a QExactive HF-Orbitrap mass spectrometer.

Figure 4. Absolute quantitation of AKT/mTOR phosphor pathway proteins in mammalian cells. MCF7 cells (human breast cancer cell line) were treated with IGF1 and the absolute quantitation of AKT/mTOR pathway proteins was determined using the SureQuant AKT Pathway Multiplex Panel (Absolute Quantitation). The pathway proteins were enriched through multiplex immunoprecipitation followed by nanoLC-PRM/MS analyses on a Q Exactive HF-Orbitrap mass spectrometer. All targets were quantified by standard curve generated for each target peptides included in the Quantitation Modules.

Graph showing absolute quantitation of AKT/mTOR pathway proteins in MCF7 cells.
Graph showing relative quantitation of AKT isoforms in A549 cells.

Figure 5. Relative quantitation of AKT isoforms in mammalian cells. A549 cells (human adenocarcinoma cell line) were treated with IGF1 and the relative quantitation of AKT isoforms was determined using the SureQuant AKT Isoform Panel (Relative Quantitation). The AKT isoforms were enriched through multiplex immunoprecipitation followed by nanoLC-PRM/MS analyses on a QExactive HF-Orbitrap mass spectrometer.

RAS isoform and TP53 reagents

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.

A.

Graph showing relative quantitation of RAS isoforms and TP53 in mammalian cells

B.

Graph showing relative quantitation of RAS isoforms and TP53 in mammalian cells

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.

Phosphopeptide and System Suitability standards

  • 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).
Chromatogram of SureQuant Phosphopeptide Suitability Standard

Figure 7. SureQuant Phosphopeptide Suitability Standard. Base peak chromatogram (top) and Skyline chromatogram (bottom) of SureQuant Phosphopeptide Suitability Standard (200 fmol) were separated using a Dionex UltiMate 3000 RSLCnano System and Thermo Scientific™ Acclaim PepMap RSLC C18 column (2 μm x 75 μm x 50 cm) with a 2–35% gradient (Buffer A: 0.1% FA in water, Buffer B: 0.1% FA in 100% acetonitrile) at 300 nL/min for 60 minutes. Detection was performed on a Thermo Scientific Q Exactive HF-Orbitrap Mass Spectrometer.

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)).

Schematic showing relative quantitation of phosphopeptides using Multipathway Phosphopeptide Standard.

A.

Schematic showing relative quantitation of phosphopeptides using Multipathway Phosphopeptide Standard.

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).
Chromatogram of Pierce LC-MS/MS System Suitability Standard (7x5)

A.

Chromatogram of Pierce LC-MS/MS System Suitability Standard (7x5)

B.

Figure 9. Pierce LC-MS/MS System Suitability Standard (7x5 mix). A. Composition of Pierce LC-MS/MS System Suitability Standard (7 x 5 mix). B. For MS analysis, the standards were analyzed on a Thermo Scientific UltiMate™ 3000 RSLCnano System and Q Exactive HF system using an EASY-Spray HPLC Column with a 3–30% gradient of Buffer B (0.1% formic acid, 99.9% acetonitrile) and Buffer A (0.1% formic acid) at
0.3 µL/min.

Videos and Webinars

與 Jonathan Krieger 針對 SureQuant 靶向质谱分析测定试剂盒的访谈

Dr. Jonathan Krieger, former Lab Research Project Manager at the SPARC Biocentre at SickKids hospital in Toronto, Canada, discusses how the SureQuant Targeted MS Assay Kits have improved his cancer research results.

Investigating the AKT-mTOR pathway with targeted mass spectrometry applications:

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.

Resources
custom-peptide-synthesis-400x350-2

Custom peptide synthesis

 Download brochure

References
Stylesheet for Classic Wide Template adjustments

仅供科研使用,不可用于诊断目的。