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SureQuant™ AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation) (Thermo Scientific™)

The Thermo Scientific SureQuant AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation) provides a complete reagent and quantitation solution for the enrichment and analysis of key phosphorylation events of AKT signaling pathway proteins using mass spectrometry (MS).

The SureQuant AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation) consists of two modules:
SureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module includes reagents necessary to immuno-enrich phosphorylated AKT-mTOR pathway proteins and perform in-solution MS sample preparation from sample lysis through peptide clean-up
SureQuant AKT Pathway (Phospho) Absolute Quantitation Module includes a system suitability standard to monitor MS performance, AQUA Ultimate HeavyPeptide and LightPeptide mixtures for absolute peptide quantitation, and methods for using Thermo Scientific Q Exactive and Fusion Tribrid instruments and Nanospray LC instrumentation

SureQuant AKT Pathway (Phospho) Multiplex Panel features include:
Complete—includes all reagents for successful monitoring of MS system performance, sample preparation, and absolute quantitation of target proteins/peptides
Validated—antibodies, peptides, and control lysate rigorously tested for specificity and successful quantitation of each target peptide
Multiplex—able to quantitate phosphorylation of ten key proteins in the AKT signaling pathway
Flexible—modular format allows for immuno-enrichment only, or in combination with relative or absolute quantitation

The AKT signaling pathway serves as a master regulatory pathway for cell growth and proliferation, and phosphorylation plays a major role in its modulation. Aberrant activation of this pathway promotes survival of tumor cells in many types of cancer. This multiplex kit is ideal for monitoring protein expression of ten key phosphorylated proteins in the AKT signaling pathway (AKT, PTEN, IRS1, IGF1-R, GSK3a, GSK3b, PRAS40, mTOR, p70S6K, and TSC2) in a single assay. The ability to simultaneously monitor and quantitate these targets in a single sample is a powerful tool for assessment of normal and abnormal pathway signaling.

The SureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module enables highly effective antigen immunoprecipitation (IP) and co-immunoprecipitation (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, with non-specific proteins removed through washing with MS-compatible buffers. The target proteins are eluted using trypsin, and are directly compatible with MS sample preparation. The MS sample preparation has been streamlined and can be completed in ~4 hours for same-day MS analysis.

The SureQuant AKT Pathway (Phospho) Absolute Quantitation Module provides reagents for absolute quantitation of 30 unique peptides from 10 phosphorylated AKT-mTOR signaling pathway target proteins using liquid chromatography (LC) and MS. AQUA Ultimate HeavyPeptide mix, AQUA Ultimate LightPeptide mix, and Pierce 6 Protein Digest, equimolar, LC-MS grade reagents are used to generate a standard curve in a matrix, and serial dilutions performed by the user have been optimized to span 2–3 orders of magnitude (200 to 0.03 fmol on column). Standardized methods and data process templates are provided for streamlined data acquisition and analysis.

The Pierce LC-MS/MS System Suitability Standard (7 x 5 Mix) is included in this module to assess dynamic range of LC-MS/MS systems before running the calibration curves and samples.

Together, these modules provide a complete solution for sample preparation and analysis of the phosphorylated target proteins in the pathway.

Related products:
EASY-Spray LC Column
SureQuant AKT Pathway Multiplex Panel (Absolute Quantitation)
Pierce Trypsin Protease, MS Grade
Pierce LC-MS/MS System Suitability Standard

SureQuant™ AKT Pathway (Phospho) Multiplex Panel (Relative Quantitation) (Thermo Scientific™)

The Thermo Scientific SureQuant AKT Pathway (Phospho) Multiplex Panel (Relative Quantitation) provides a complete reagent and quantitation solution for the enrichment and analysis of key phosphorylation events of AKT signaling pathway proteins using mass spectrometry (MS).

The SureQuant AKT Pathway (Phospho) Multiplex Panel (Relative Quantitation) consists of two modules:
SureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module includes reagents necessary to immuno-enrich phosphorylated AKT-mTOR pathway proteins and perform in-solution MS sample preparation from sample lysis through peptide clean-up
SureQuant AKT Pathway (Phospho) Relative Quantitation Module includes a system suitability standard to monitor MS performance, AQUA Ultimate HeavyPeptide mix for relative peptide quantitation, and methods for using Thermo Scientific Q Exactive and Fusion Tribrid instruments and Nanospray LC instrumentation

SureQuant AKT Pathway (Phospho) Multiplex Panel features include:
Complete—includes all reagents for successful monitoring of MS system performance, sample preparation, and absolute quantitation of target proteins/peptides
Validated—antibodies, peptides, and control lysate rigorously tested for specificity and successful quantitation of each target peptide
Multiplex—able to quantitate phosphorylation of ten key proteins in the AKT signaling pathway
Flexible—modular format allows for immuno-enrichment only, or in combination with relative or absolute quantitation

The AKT signaling pathway serves as a master regulatory pathway for cell growth and proliferation, and phosphorylation plays a major role in its modulation. Aberrant activation of this pathway promotes survival of tumor cells in many types of cancer. This multiplex kit is ideal for monitoring protein expression of ten key phosphorylated proteins in the AKT signaling pathway (AKT, PTEN, IRS1, IGF1-R, GSK3a, GSK3b, PRAS40, mTOR, p70S6K, and TSC2) in a single assay. The ability to simultaneously monitor and quantitate these targets in a single sample is a powerful tool for assessment of normal and abnormal pathway signaling.

The SureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module enables highly effective antigen immunoprecipitation (IP) and co-immunoprecipitation (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, with non-specific proteins removed through washing with MS-compatible buffers. The target proteins are eluted using trypsin, and are directly compatible with MS sample preparation. The MS sample preparation has been streamlined and can be completed in ~4 hours for same-day MS analysis.

The SureQuant AKT Pathway (Phospho) Relative Quantitation Module provides reagents for relative quantitation of 30 unique peptides from 10 phosphorylated AKT-mTOR signaling pathway target proteins using liquid chromatography (LC) and MS. AQUA Ultimate HeavyPeptide mixture is provided for use as a spike-in internal standard for relative quantitation in the multiplexed sample. Standardized methods and data process templates are provided for streamlined data acquisition and analysis.

The Pierce LC-MS/MS System Suitability Standard (7 x 5 Mix) is included in this module to assess dynamic range of LC-MS/MS systems before running the calibration curves and samples.

Together, these modules provide a complete solution for sample preparation and analysis of the phosphorylated target proteins in the pathway.

Related products:
EASY-Spray LC Column
SureQuant AKT Pathway Multiplex Panel (Absolute Quantitation)
Pierce Trypsin Protease, MS Grade
Pierce LC-MS/MS System Suitability Standard

SureQuant™ AKT Pathway (Phospho) IP and MS Sample Preparation Module (Thermo Scientific™)

The Thermo Scientific SureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module provides a complete reagent solution for the multiplexed enrichment and sample preparation for studies of key phosphorylation events of the AKT signaling pathway using mass spectrometry (MS).

SureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module features include:
• Complete—includes all of the reagents necessary for sample lysis, immuno-enrichment, and MS sample preparation
• Validated—antibodies validated for specificity and background
• Optimized—procedure simplified and condensed to prepare samples after immunoprecipitation in four hours
• Multiplex—able to immuno-enrich phosphorylated AKT, PTEN, IRS1, IGF1-R, GSK3a, GSK3b, PRAS40, mTOR, p70S6K, and TSC2 in a single reaction

The AKT signaling pathway serves as a master regulatory pathway for cell growth and proliferation, and phosphorylation plays a major role in its modulation. Aberrant activation of this pathway promotes survival of tumor cells in many types of cancer. This module is ideal for the multiplexed immuno-enrichment of key phosphorylated proteins in the AKT signaling pathway and has been optimized for use with the SureQuant quantitation modules.

The SureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module enables highly effective antigen immunoprecipitation (IP) and co-immunoprecipitation (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, with non-specific proteins removed through washing with MS-compatible buffers. The target proteins are eluted using trypsin and are directly compatible with MS sample preparation. The MS sample preparation has been streamlined and can be completed in ~4 hours for same-day MS analysis.

This kit can be used with the either the SureQuant AKT Pathway (Phospho) Absolute Quantitation Module or the SureQuant AKT Pathway (Phospho) Relative Quantitation Module for protein targeted quantitation. When used together, these modules provide a complete solution for sample preparation and analysis of the phosphorylated target proteins in the pathway.

Related products:
EASY-Spray LC Column
SureQuant AKT Pathway Multiplex Panel (Absolute Quantitation)
Pierce Trypsin Protease, MS Grade
Pierce LC-MS/MS System Suitability Standard

SureQuant™ AKT Pathway (Phospho) Relative Quantitation Module (Thermo Scientific™)

The Thermo Scientific SureQuant AKT Pathway (Phospho) Relative Quantitation Module provides a complete quantitation solution for the analysis of key phosphorylation events of AKT signaling pathway proteins using mass spectrometry (MS).

SureQuant AKT Pathway (Phospho) Relative Quantitation Module features include:
Complete—contains validated AQUA HeavyPeptide mix, diluent, matrix, system suitability standard including optimized acquisition and analysis methods
Validated—peptides rigorously tested for successful quantitation of each target peptide
Multiplex—able to quantitate phosphorylated AKT, PTEN, IRS1, IGF1-R, GSK3a, GSK3b, PRAS40, mTOR, p70S6K, and TSC2 in a single reaction

The AKT signaling pathway serves as a master regulatory pathway for cell growth and proliferation, and phosphorylation plays a major role in its modulation. Aberrant activation of this pathway promotes survival of tumor cells in many types of cancer. This multiplex kit is ideal for monitoring protein expression of ten key phosphorylated proteins in the AKT signaling pathway (AKT, PTEN, IRS1, IGF1-R, GSK3a, GSK3b, PRAS40, mTOR, p70S6K, and TSC2) in a single assay. The ability to simultaneously monitor and quantitate these targets in a single sample is a powerful tool for assessment of normal and abnormal pathway signaling.

The SureQuant AKT Pathway (Phospho) Relative Quantitation Module provides reagents for relative quantitation of 30 unique peptides from 10 phosphorylated AKT-mTOR signaling pathway target proteins using liquid chromatography (LC) and MS. AQUA Ultimate HeavyPeptide mixture is provided for use as a spike-in internal standard for relative quantitation in the multiplexed sample. Standardized methods and data process templates are provided for streamlined data acquisition and analysis.

The Pierce LC-MS/MS System Suitability Standard (7 x 5 Mix) is included in this module to assess dynamic range of LC-MS/MS systems before running the calibration curves and samples.

This kit can be used with the SureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module for enriched multiplex sample preparation. When used together, these modules provide a complete solution for sample preparation and analysis of the phosphorylated target proteins in the pathway.

Related products:
EASY-Spray LC Column
SureQuant AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation)
Pierce Trypsin Protease, MS Grade
Pierce LC-MS/MS System Suitability Standard

SureQuant™ AKT Pathway (Phospho) Absolute Quantitation Module (Thermo Scientific™)

The Thermo Scientific SureQuant AKT Pathway (Phospho) Absolute Quantitation Module provides a complete quantitation solution for the analysis of key phosphorylation events of AKT signaling pathway proteins using mass spectrometry (MS).

SureQuant AKT Pathway (Phospho) Absolute Quantitation Module features include:
Complete—contains validated AQUA HeavyPeptide and LightPeptide mixtures, diluent, matrix, system suitability standard including optimized acquisition and analysis methods
Validated—peptides rigorously tested for successful quantitation of each target peptide
Multiplex—able to quantitate phosphorylated AKT, PTEN, IRS1, IGF1-R, GSK3a, GSK3b, PRAS40, mTOR, p70S6K, and TSC2 in a single reaction

The AKT signaling pathway serves as a master regulatory pathway for cell growth and proliferation, and phosphorylation plays a major role in its modulation. Aberrant activation of this pathway promotes survival of tumor cells in many types of cancer. This multiplex kit is ideal for monitoring protein expression of ten key phosphorylated proteins in the AKT signaling pathway (AKT, PTEN, IRS1, IGF1-R, GSK3a, GSK3b, PRAS40, mTOR, p70S6K, and TSC2) in a single assay. The ability to simultaneously monitor and quantitate these targets in a single sample is a powerful tool for assessment of normal and abnormal pathway signaling.

The SureQuant AKT Pathway (Phospho) Absolute Quantitation Module provides reagents for absolute quantitation of 30 unique peptides from 10 phosphorylated AKT-mTOR signaling pathway target proteins using liquid chromatography (LC) and MS. AQUA Ultimate HeavyPeptide mix, AQUA Ultimate LightPeptide mix, and Pierce 6 Protein Digest, equimolar, LC-MS grade reagents are used to generate a standard curve in a matrix, and serial dilutions performed by the user have been optimized to span 2–3 orders of magnitude (200 to 0.03 fmol on column). Standardized methods and data process templates are provided for streamlined data acquisition and analysis.

The Pierce LC-MS/MS System Suitability Standard (7 x 5 Mix) is included in this module to assess dynamic range of LC-MS/MS systems before running the calibration curves and samples.

This kit can be used with the SureQuant AKT Pathway (Phospho) IP and MS Sample Preparation Module for enriched multiplex sample preparation. When used together, these modules provide a complete solution for sample preparation and analysis of the phosphorylated target proteins in the pathway.

Related products:
EASY-Spray LC Column
SureQuant AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation)
Pierce Trypsin Protease, MS Grade
Pierce LC-MS/MS System Suitability Standard

SureQuant™ AKT Isoform Panel (Absolute Quantitation) (Thermo Scientific™)

The Thermo Scientific SureQuant AKT Isoform Panel (Absolute Quantitation) provides a complete reagent and quantitation solution for the enrichment and analysis of AKT1, AKT2, and AKT3 isoforms using mass spectrometry (MS).

The SureQuant AKT Isoform Panel (Absolute Quantitation) consists of two modules:
SureQuant AKT Isoform IP and MS Sample Preparation Module includes all of the reagents necessary to immuno-enrich AKT1, AKT2, and AKT3 proteins and perform in-solution MS sample preparation from sample lysis through peptide clean-up
SureQuant AKT Isoform Absolute Quantitation Module includes a system suitability standard to monitor MS performance, AQUA Ultimate HeavyPeptide and LightPeptide mixtures for absolute peptide quantitation, and methods for using Thermo Scientific Q Exactive and Fusion Tribrid instruments and Nanospray LC instrumentation

SureQuant AKT Isoform Panel features include:
Complete—includes all reagents for successful monitoring of MS system performance, sample preparation, and absolute quantitation of target proteins/peptides
Validated—antibodies, peptides, and control lysate rigorously tested for specificity and successful quantitation of each target peptide
Multiplex—able to quantitate isoforms and phosphorylation of AKT
Flexible—modular format allows for immuno-enrichment only, or in combination with relative or absolute quantitation

The AKT signaling pathway serves as a master regulatory pathway for cell growth and proliferation, and AKT plays a vital role in the signaling cascade. Aberrant activation of this pathway promotes survival of tumor cells in many types of cancer. This kit is ideal for monitoring AKT expression levels of the three 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 pathway signaling.

The SureQuant AKT Isoform IP and MS Sample Preparation Module enables highly effective antigen immunoprecipitation (IP) and co-immunoprecipitation (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, where non-specific proteins are removed through washing with MS-compatible buffers. The target proteins are eluted using trypsin and are directly compatible with MS sample preparation. The MS sample preparation has been streamlined and can be completed in ~4 hours for same-day MS analysis.

The SureQuant AKT Isoform Absolute Quantitation Module provides reagents for absolute quantitation of six unique peptides from AKT1, AKT2, and AKT3 isoforms using liquid chromatography (LC) and MS. AQUA Ultimate HeavyPeptide mix, AQUA Ultimate LightPeptide mix, and Pierce 6 Protein Digest, equimolar, LC-MS grade reagents are used to generate a standard curve in a matrix, and serial dilutions performed by the user have been optimized to span 2–3 orders of magnitude (200 0.03 fmol on column). Standardized methods and data process templates are provided for streamlined data acquisition and analysis.

The Pierce LC-MS/MS System Suitability Standard (7 x 5 Mix) is included in this module to assess dynamic range of LC-MS/MS systems before running the calibration curves and samples.

Together, these modules provide a complete solution for sample preparation and analysis of the target proteins.

Related products:
EASY-Spray LC Column
SureQuant AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation)
Pierce Trypsin Protease, MS Grade
Pierce LC-MS/MS System Suitability Standard

SureQuant™ AKT Isoform Panel (Relative Quantitation) (Thermo Scientific™)

The Thermo Scientific SureQuant AKT Isoform Panel (Relative Quantitation) provides a complete reagent and quantitation solution for the enrichment and analysis of AKT1, AKT2, and AKT3 isoforms using mass spectrometry (MS).

The SureQuant AKT Isoform Panel (Relative Quantitation) consists of two modules:
SureQuant AKT Isoform IP and MS Sample Preparation Module includes all of the reagents necessary to immuno-enrich AKT1, AKT2, and AKT3 proteins and perform in-solution MS sample preparation from sample lysis through peptide clean-up
SureQuant AKT Isoform Relative Quantitation Module includes a system suitability standard to monitor MS performance, AQUA Ultimate HeavyPeptide mixture for relative peptide quantitation, and methods for using Thermo Scientific Q Exactive and Fusion Tribrid instruments and Nanospray LC instrumentation

SureQuant AKT Isoform Panel features include:
Complete—includes all reagents for successful monitoring of MS system performance, sample preparation, and absolute quantitation of target proteins/peptides
Validated—antibodies, peptides, and control lysate rigorously tested for specificity and successful quantitation of each target peptide
Multiplex—able to quantitate isoforms and phosphorylation of AKT
Flexible—modular format allows for immuno-enrichment only, or in combination with relative or absolute quantitation

The AKT signaling pathway serves as a master regulatory pathway for cell growth and proliferation, and AKT plays a vital role in the signaling cascade. Aberrant activation of this pathway promotes survival of tumor cells in many types of cancer. This kit is ideal for monitoring AKT expression levels of the three 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 pathway signaling.

The SureQuant AKT Isoform IP and MS Sample Preparation Module enables highly effective antigen immunoprecipitation (IP) and co-immunoprecipitation (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, where non-specific proteins are removed through washing with MS-compatible buffers. The target proteins are eluted using trypsin and are directly compatible with MS sample preparation. The MS sample preparation has been streamlined and can be completed in ~4 hours for same-day MS analysis.

The SureQuant AKT Isoform Relative Quantitation Module provides reagents for relative quantitation of six unique peptides from AKT1, AKT2, and AKT3 isoforms using liquid chromatography (LC) and MS. AQUA Ultimate HeavyPeptide mixture is provided for use as a spike-in internal standard for relative quantitation in the multiplexed sample. Standardized methods and data process templates are provided for streamlined data acquisition and analysis.

The Pierce LC-MS/MS System Suitability Standard (7 x 5 Mix) is included in this module to assess dynamic range of LC-MS/MS systems before running the calibration curves and samples.

Together, these modules provide a complete solution for sample preparation and analysis of the target proteins.

Related products:
EASY-Spray LC Column
SureQuant AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation)
Pierce Trypsin Protease, MS Grade
Pierce LC-MS/MS System Suitability Standard

MSIA™ D.A.R.T.'S™ for the Versette and Novus i Platform (Thermo Scientific™)

Improve hybrid MS bio-analysis with Thermo Scientific™ MSIA™ D.A.R.T.'S™ for the Versette and Novus i Platform an exclusive Thermo Fisher Scientific workflow solution for proteins and peptides.

HCT 116 (+hIGF-1) Control Lysate (1mg/mL) (Thermo Scientific™)

Thermo Scientific HCT 116 (+hIGF-1) Control Lysate is a stimulated cell lysate that has been validated for the expression levels and phosphorylation of key targets in the RAS signaling pathway.

HCT 116 (+hIGF-1) Control Lysate features include:
• Validated for the expression RAS by western blotting and of NRAS, KRAS, and HRAS by mass spectrometry analysis
• Optimized—stimulation and lysis conditions optimized for detection of RAS signaling targets
• Convenient—lysate is ready-to-use for immunoprecipitation, immunoprecipitation to mass spectrometry, and western blotting applications
• Stable for two years at -80°C

The RAS family of GTPase proteins modulates two key signaling pathways involved in cell growth and proliferation: the MAPK and PI3K pathways. Aberrant activation of RAS proteins promotes constitutive activation of cell growth and proliferation that can result in cancer. Mutations in the NRAS, KRAS, and HRAS oncogenes are the most common mutations in human cancer. This lysate has been validated for expression levels of the three isoforms of RAS using mass spectrometry.

The HCT 116 cell line is a human colon cancer epithelial cell line and has been used for proliferation and tumorigenicity studies for colon cancer. The HCT 116 (+hIGF-1) cell lysate is provided as frozen lysate at a standard concentration of 1 mg/mL (500 mg per vial). Lysates are prepared in non-denaturing buffer without loading dyes or dissociating agents (i.e., no 2-mercaptoethanol or dithiothreitol has been added). For western blot analysis, heat lysate to 95°C for 5 minutes. Add reducing agent before heating if desired. The recommended loading volume per lane is 10–20 μL (10–20 µg).

Related products
SureQuant AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation)
SureQuant RAS Isoform Panel (Absolute Quantitation)
SureQuant TP53 Panel (Absolute Quantitation)

MSIA™ Microcolumns for the Freedom EVO Platform (Thermo Scientific™)

Next generation large molecule bio-analysis workflow for Freedom EVO Platform .

High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Scientific™)

The Thermo Scientific™ High-Select™ TiO2 Phosphopeptide Enrichment Kit enables efficient enrichment of phosphorylated peptides from complex and fractionated protein digests for analysis by mass spectrometry (MS). (This product replaces Cat. No. 88301.)

Features of the TiO2 Phosphopeptide Enrichment Kit:
• Convenient—pre-formulated buffers for ease of use and spin-tip format to enable parallel processing of multiple samples
• High binding capacity—each column enriches up to 150 μg of phosphopeptides
• High specificity—recovers phosphopeptides with > 90% selectivity
• Complementary—TiO2 enriches a unique set of phosphopeptides that complements the High-Select™ Fe-NTA IMAC Phosphopeptide Enrichment Kit

The complete kit includes 24 titanium dioxide (TiO2) spin tips with optimized buffers to facilitate preparation of enriched phosphopeptides. Each column can enrich phosphopeptides from 0.5 to 3 mg of total protein digest as starting sample. The optimized buffers and simplified procedure eliminate the need for pyrrolidine in the elution buffer and subsequent clean-up using graphite spin columns. Use of this kit results in enrichment of up to 150 µg of phosphopeptides with greater than 90% selectivity.

Mass spectrometry is a key tool for identifying sites of protein phosphorylation and quantifying phosphorylation changes; however, MS analysis of protein phosphorylation is challenging due to the low stoichiometry, high hydrophilicity, poor ionization, and incomplete fragmentation of phosphopeptides. Because of the low relative abundance of phosphorylation modifications in complex protein samples, enrichment is essential for successful MS analysis of phosphopeptides. The improved High-Select TiO2 Phosphopeptide Enrichment Kit complements our lysis, reduction, alkylation, and digestion reagents, along with our C18, graphite spin, and high-pH reversed-phase peptide fractionation columns to provide a complete workflow for phosphopeptide enrichment.

The new procedure and optimized buffers have been shown to maintain the excellent selectivity and yield of the previous kit (Figure 1), while removing hazardous reagents and clean-up. The High-Select TiO2 Phosphopeptide Enrichment Kit is complementary to the High-Select Fe-NTA Phosphopeptide Enrichment Kit, with both kits enriching similar numbers of phosphopeptides but different subpopulations of phosphorylated peptides (Figure 2).

For high resolution analysis of the enriched phosphopeptides, the recommended LC column for Nanospray Flex source is Acclaim PepMap 100 C18 LC Column (Cat. No. 164942 or 164939). For EASY-Spray source, the recommended LC column is EASY-Spray C18 LC Column (Cat. No. ES802 or ES803).

SureQuant™ TP53 Panel (Absolute Quantitation) (Thermo Scientific™)

The Thermo Scientific SureQuant TP53 Panel (Absolute Quantitation) provides a complete reagent and quantitation solution for the enrichment and analysis of TP53 expression and phosphorylation using mass spectrometry (MS).

The SureQuant TP53 Panel (Absolute Quantitation) consists of two modules:
SureQuant TP53 Panel IP and MS Sample Preparation Module includes all of the reagents necessary to immuno-enrich TP53 and perform in-solution MS sample preparation from sample lysis through peptide clean-up
SureQuant TP53 Panel Absolute Quantitation Module includes a system suitability standard to monitor MS performance, AQUA Ultimate HeavyPeptide and LightPeptide mixtures for absolute peptide quantitation, and methods for using Thermo Scientific Q Exactive and Fusion Tribrid instruments and Nanospray LC instrumentation

SureQuant TP53 Panel features include:
Complete—includes all reagents for successful monitoring of MS system performance, sample preparation, and absolute quantitation of target proteins/peptides
Validated—antibodies, peptides, and control lysate rigorously tested for specificity and successful quantitation of each target peptide
Multiplex—able to quantitate expression and phosphorylation of TP53
Flexible—modular format allows for immuno-enrichment only, or in combination with relative or absolute quantitation

The tumor suppressor protein TP53 functions as a negative checkpoint regulator for abnormal cell signaling and can induce cell cycle arrest, activate DNA repair proteins, or initiate apoptosis. Mutations in TP53 often result abnormal cell growth and are commonly found in many types of cancer. This kit is ideal for monitoring TP53 expression 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 pathway signaling.

The SureQuant TP53 Panel IP and MS Sample Preparation Module enables highly effective antigen immunoprecipitation (IP) and co-immunoprecipitation (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, where non-specific proteins are removed through washing with MS-compatible buffers. The target proteins are eluted using trypsin and are directly compatible with MS sample preparation. The MS sample preparation has been streamlined and can be completed in ~4 hours for same-day MS analysis.

The SureQuant TP53 Panel Absolute Quantitation Module provides reagents for absolute quantitation of four unique peptides from TP53 using liquid chromatography (LC) and MS. AQUA Ultimate HeavyPeptide mix, AQUA Ultimate LightPeptide mix, and Pierce 6 Protein Digest, equimolar, LC-MS grade reagents are used to generate a standard curve in a matrix, and serial dilutions performed by the user have been optimized to span 2–3 orders of magnitude (200 to 0.03 fmol on column). Standardized methods and data process templates are provided for streamlined data acquisition and analysis.

The Pierce LC-MS/MS System Suitability Standard (7 x 5 Mix) is included in this module to assess dynamic range of LC-MS/MS systems before running the calibration curves and samples.

Together, these modules provide a complete solution for sample preparation and analysis of the target proteins.

Related products:
EASY-Spray LC Column
SureQuant AKT Pathway (Phospho) Multiplex Panel (Absolute Quantitation)
Pierce Trypsin Protease, MS Grade
Pierce LC-MS/MS System Suitability Standard

SureQuant™ AKT Isoform IP and MS Sample Preparation Module (Thermo Scientific™)

The Thermo Scientific SureQuant AKT Isoform IP and MS Sample Preparation Module provides a complete reagent solution for the multiplexed enrichment and sample preparation of AKT1, AKT2, and AKT3 for mass spectrometry (MS).

SureQuant AKT Isoform IP and MS Sample Preparation Module features include:
Complete—includes all of the reagents necessary for sample lysis, immuno-enrichment, and MS sample preparation
Validated—antibodies validated for specificity and background
Optimized—procedure simplified and condensed to prepare samples after immunoprecipitation in four hours
Multiplex—able to immuno-enrich AKT1, AKT2, and AKT3 isoforms in a single reaction

The AKT signaling pathway serves as a master regulatory pathway for cell growth and proliferation, and AKT plays a vital role in the signaling cascade. Aberrant activation of this pathway promotes survival of tumor cells in many types of cancer. This module is ideal for immuno-enrichment of the AKT1, AKT2, and AKT3 isoforms for use with the SureQuant quantitation modules.

The SureQuant AKT Isoform IP and MS Sample Preparation Module enables highly effective antigen immunoprecipitation (IP) and co-immunoprecipitation (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, where non-specific proteins are removed through washing with MS-compatible buffers. The target proteins are eluted using trypsin and are directly compatible with MS sample preparation. The MS sample preparation has been streamlined and can be completed in ~4 hours for same-day MS analysis.

This kit can be used with the either the SureQuant AKT Isoform Absolute Quantitation Module or the SureQuant AKT Isoform Relative Quantitation Module for protein targeted quantitation. When used together, these modules provide a complete solution for sample preparation and analysis of the target proteins.

Related products:
EASY-Spray LC Column
SureQuant AKT Pathway Multiplex Panel (Absolute Quantitation)
Pierce Trypsin Protease, MS Grade
Pierce LC-MS/MS System Suitability Standard

Pierce™ Lys-C Protease, MS Grade (Thermo Scientific™)

Thermo Scientific Lys-C Protease, MS Grade, is a highly purified native endoproteinase validated for maximum activity and stability in proteomic applications.

Features of MS Grade Lys-C Protease:

• Enhanced digestion—use in tandem with trypsin to decrease tryptic missed cleavages
• Increased sequence coverage—better protein characterization results from overlapping peptides with complementary chromatographic, ionization and fragmentation properties
• Versatile—effective enzyme activity under highly denaturing conditions (e.g., 8M urea)
• C-terminal lysine cleavage specificity—at least 90% for a complex protein sample
• Stable—provided in a lyophilized format

Product Details
This Lys-C is a mass spectrometry (MS)-grade serine protease isolated from Lysobacter enzymogenes. Lys-C has high activity and specificity for lysine residues resulting in larger peptides and less sample complexity than trypsin (i.e. fewer peptides). Unlike trypsin, Lys-C can cleave lysines followed by prolines, making it ideal for sequential protein digestion followed by trypsin to decrease missed cleavages. These unique Lys-C properties ensure high digestion efficiency when used alone or followed by tryptic digestion. Additionally, Lys-C prototypic peptides typically have higher charge states, making it an enzyme of choice for use with ETD fragmentation. Lys-C is commonly in phosphopeptide enrichment workflows because it generates peptides with primary amines at both the N-and C-terminus allowing the fragments to be double-labeled with amine-reactive isobaric tags. This results in enhanced peptide ionization and improved limits of quantitation since more fragment ions can be re-isolated during MS3 acquisition. This enzyme can be used for in-solution or in-gel digestion workflows to produce peptides for LC-MS/MS protein identification. This Lys-C enzyme is packaged lyophilized (20 µg or 100 µg quantities).

The endoproteinase LysC specifically hydrolyzes proteins at the carboxyl side of lysine. Efficient protein digestion can be completed in 2 hours at 37°C. LysC remains active in highly denaturing conditions such as 8M urea, 2M guanidine·HCl, 1% SDS, 2% CHAPS and 40% acetonitrile and functions well within pH 7-9 (maximal activity at pH 8). This lyophilized enzyme has a mass of 30 kDa and is stable for 1 year when stored at -20°C.

Applications
• Improved sequence coverage of protein digests
De novo sequencing
• Epigenetic studies
• In-gel and in-solution digestion of proteins

High Select™ Top14 Abundant Protein Depletion Resin (Thermo Scientific™)

Thermo Scientific High Select Top14 Abundant Protein Depletion Resin decreases the abundant protein and antibody components of human plasma samples, helping prepare the samples for mass spectrometry or 1D or 2D electrophoresis.

Features of Top14 Abundant Protein Depletion Resin include:
• Flexible—provided in bulk format for depletion of user-defined amount of human plasma sample
• Optimized—remove >95% of IgG and >95% of albumin, plus 12 other abundant proteins (Top 14)
• Fast—process samples in ~10 minutes
• Economical—cost-effective bulk format

High Select Top14 Abundant Protein Depletion Resin uses highly specific immobilized antibodies to remove human serum albumin (HSA), albumin, IgG, IgA, IgM, IgD, IgE, kappa and lambda light chains, alpha-1-acidglycoprotein, alpha-1-antitrypsin, alpha-2-macroglobulin, apolipoprotein A1, fibrinogen, haptoglobin, and transferrin from serum, plasma, or spinal fluids. The resin is provided in an economical bulk format that may be used to fill columns or plates, depending on sample volumes.

Analysis of human fluids is often complicated by the presence of high concentrations of albumin and IgG that can make up more than 70% of total serum protein. Removal of these proteins is often essential for the study of low-abundant proteins. High Select Top14 Abundant Protein Depletion Resin can deplete greater than 95% of the 14 most abundant proteins in serum. Columns or plates are filled with the resin based on the needs of the user, and samples processed in approximately 10 minutes. The depletion of highly abundant proteins enables the detection of low abundant proteins in samples and subsequent identification by mass spectrometry or 1D or 2D gel electrophoresis.