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SureQuant™ RAS Isoform Relative Quantitation Module (Thermo Scientific™)

The Thermo Scientific SureQuant RAS Isoform Relative Quantitation Module provides a complete quantitation solution for the analysis of key RAS expression using mass spectrometry (MS).

SureQuant RAS Isoform Relative Quantitation Module features include:
Complete—contains validated AQUA HeavyPeptide mix, diluent, matrix, and system suitability standard including optimized acquisition and analysis methods
Validated—peptides rigorously tested for successful quantitation of each target peptide
Multiplex—able to quantitate NRAS, KRAS, and HRAS isoforms

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 the 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 kit is ideal for monitoring expression levels of the three isoforms 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 RAS Isoform Relative Quantitation Module provides reagents for relative quantitation of three unique peptides from RAS 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 SureQuant RAS Isoform 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 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

Pierce™ C18 Tips, 10 µL bed (Thermo Scientific™)

Thermo Scientific Pierce C18 Tips are ready-to-use pipette-tip columns of C18 resin that enable fast and efficient capture, concentration, desalting and elution of peptides for MALDI mass spectrometry and other methods.

Improve protein analysis results with Pierce C18 Tips by removing urea, salts and other contaminants before MS analysis. The tips are ideal for preparing samples for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization techniques. The Pierce C18 Tips are available in convenient 10 and 100 µL tips with binding capacities of 8 and 80 µg, respectively.

Features of C18 Tips:

• Monolithic C18 fast-flow tips have low resistance and improved flow characteristics compared to other commercially available tips
• High binding capacity in a convenient tip format
• Two pipette tip formats for processing up to 8 or 80 µg of sample
• Eight packs and 96-tip trays are automation compatible

These C18 tips offer excellent flow properties with a high-efficiency monolithic C18 sorbent for fast wetting, loading, washing and eluting. The five-step procedure is simple and requires less than 5 minutes to process protein digests, strong cation exchange fractions and other protein and peptide samples for mass spectrometric (MS) analyses.

Related Products
Pierce™ C18 Tips, 100 µL bed

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

The Thermo Scientific SureQuant AKT Isoform Absolute Quantitation Module provides a complete quantitation solution for the analysis of key AKT expression using mass spectrometry (MS).

SureQuant AKT Isoform 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 AKT1, AKT2, and AKT3 isoforms and phosphorylation of AKT

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 Absolute Quantitation Module provides reagents for absolute quantitation of six unique peptides from AKT 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 SureQuant AKT Isoform 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 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

Pierce™ Protein Precipitation Plates (Thermo Scientific™)

Thermo Scientific Pierce Protein Precipitation Plates provide an easy, 96-well format for simple, rapid and automatable precipitation and removal of proteins from plasma, serum and other biological fluid samples.

Features of Protein Precipitation Plates:

• Prepare small molecule samples for HPLC or LC-MS in 10-30 minutes
• 96-well format for automatable protein precipitation and filtration
• Optimized for parallel processing and automation
• Works seamlessly with the Rapid Equilibrium Device (RED) for plasma protein-binding assays
• No acetonitrile leakage for up to four hours
• Leach-free graded hydrophobic frit prevents blockage
• Filtrates are easily collected by vacuum, positive pressure or centrifugation
• Compatible with most organic solvents

These 96-well filtration plates are designed specifically for solvent-based (e.g., acetonitrile) precipitation of proteins and their subsequent removal from fluid samples. The plates are leak-free and retain organic solvent without dripping, even while mixing. Samples are rapidly recovered by applying vacuum or other pressure. The 2 mL well volume allows processing sample volumes of 15 to 400 mL per well, using the recommended precipitation conditions of 3:1 (v/v) solvent-to-serum ratio. The Protein Precipitation Plates are the ideal choice for downstream processing of plasma protein binding assay samples prepared with equilibrium dialysis systems.

The design of Pierce Protein Precipitation Plates provides many advantages over standard protein precipitation methods based solely on either filtration or centrifugation. The plates use a special graded filter system that retains precipitated protein particles without clogging, yet freely allows soluble (nonprecipitated) eluates through the 0.2 µM cut-off membrane. This design maximizes protein removal and ensures the highest possible level of sample recovery.

The study of small molecules and how they behave in complex biofluids (serum, plasma, blood and urine) is an essential part of pharmaceutical, DMPK and metabolomics research. Drugs or other small molecules are routinely monitored for clearance rates, steady-state concentrations, serum-binding equilibrium or in vivo modification. These studies require isolation of target molecules from the protein matrix before analysis. Precipitating proteins with acetonitrile and then removing the precipitate by centrifugation or filtration is the typical isolation process. Although centrifugation to form pellets is inexpensive and reliable, 96-well filtration plates provide an easy format for reproducible, simple, rapid and automatable protein precipitation and sample clarification.

SureQuant™ RAS Isoform Absolute Quantitation Module (Thermo Scientific™)

The Thermo Scientific SureQuant RAS Isoform Absolute Quantitation Module provides a complete quantitation solution for the analysis of key RAS expression using mass spectrometry (MS).

SureQuant RAS Isoform Absolute Quantitation Module features include:
Complete—contains validated AQUA HeavyPeptide and LightPeptide mixtures, diluent, matrix, and system suitability standard including optimized acquisition and analysis methods
Validated—peptides rigorously tested for successful quantitation of each target peptide
Multiplex—able to quantitate NRAS, KRAS, and HRAS isoforms

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 the 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 kit is ideal for monitoring expression levels of the three isoforms 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 RAS Isoform Absolute Quantitation Module provides reagents for absolute quantitation of three unique peptides from RAS 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 SureQuant RAS Isoform 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 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

Pierce™ C18 Tips, 100 µL bed (Thermo Scientific™)

Thermo Scientific Pierce C18 Tips are ready-to-use pipette-tip columns of C18 resin that enable fast and efficient capture, concentration, desalting and elution of peptides for MALDI mass spectrometry and other methods.

Improve protein analysis results with Pierce C18 Tips by removing urea, salts and other contaminants before MS analysis. The tips are ideal for preparing samples for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization techniques. The Pierce C18 Tips are available in convenient 10 and 100 µL tips with binding capacities of 8 and 80 µg, respectively.

Features of C18 Tips:

• Monolithic C18 fast-flow tips have low resistance and improved flow characteristics compared to other commercially available tips
• High binding capacity in a convenient tip format
• Two pipette tip formats for processing up to 8 or 80 µg of sample
• Eight packs and 96-tip trays are automation compatible

These C18 tips offer excellent flow properties with a high-efficiency monolithic C18 sorbent for fast wetting, loading, washing and eluting. The five-step procedure is simple and requires less than 5 minutes to process protein digests, strong cation exchange fractions and other protein and peptide samples for mass spectrometric (MS) analyses.

Related Products
Pierce™ C18 Tips, 10 µL bed

Pierce™ LysN Protease, MS Grade (Thermo Scientific™)

Thermo Scientific Pierce LysN Protease, MS Grade, is highly purified native Lys-N protease that has been validated for maximum activity and stability in proteomic applications.

Features of LysN Protease, MS Grade:

Thermostable – provides better digestion at higher temperatures (e.g., 50°C) in less time
Complementary to tryptic digests cleaves the opposite side of lysine from trypsin
High purity – no additional (off-target) protease activity detected
N-terminal lysine cleavage specificity – at least 90% for a complex protein sample
Versatile – enzyme is effective at many temperatures and in denaturing conditions
Stable – provided in a lyophilized format

Thermo Scientific Pierce LysN is a mass spectrometry (MS)-grade zinc metalloprotease derived from Grifola frondosa. The native Pierce LysN Protease has been highly purified to improve stability, specific activity, and cleavage selectivity. Unlike trypsin, LysN protease cleaves at the amino-terminus of lysine residues. As a result, the peptides generated by LysN are longer than those generated by trypsin and have more prevalent charged amino terminal peptide fragments. Additionally, LysN protease is more promising for epigenetic MS applications than LysC or trypsin because it is capable of cleaving methylated lysines [1]. The thermostability and chemical compatibility with denaturants make LysN protease ideal for digestion of complex protein samples for “shotgun” proteomics.

Applications:
• Improved sequence coverage of protein digests
• De novo sequencing with collision induced dissociation (CID)
• Epigenetic studies
• In-solution digestion of proteins

LysN protease is active over a wide range of temperatures and denaturing conditions. Efficient protein digestion can be completed in 2 hours at 50°C or 4 hours at 37°C, however, digestion is possible at both lower and higher temperatures. LysN also remains active under moderate denaturing conditions including 0.1% SDS, 6M urea or heating to 70°C. Maximal LysN activity occurs at pH 7 to 9. The lyophilized enzyme is stable for 2 years when stored at -20°C, and reconstituted stock solutions of LysN are stable at -80°C for 2 years or -20°C for one year without significant loss in activity.

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.

DSSO (disuccinimidyl sulfoxide) (Thermo Scientific™)

DSSO (disuccinimidyl sulfoxide) is an MS-cleavable and membrane-permeable crosslinker that contains an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of a 7-carbon spacer arm. It facilitates analysis of protein structure and complex interactions using mass spectrometry. DSSO has similar reactivity to DSS and BS3, but contains a linker that can be cleaved in the gas phase during tandem MS (MS/MS) using collision-induced dissociation (CID). The ability to cleave cross-linked peptides during MS/MS enables MS3 acquisition methods, which facilitate peptide sequencing using traditional database search engines. The MS cleavage of DSSO also generates diagnostic ion doublets during MS2, which enables identification of cross-linked peptides from dead-end modifications and searching using novel database search engines such as XlinkX.

Features of DSSO:
• Reactive groups: NHS ester (both ends)
• Reactive towards: amino groups (primary amines)
• Amine-reactive NHS ester reacts rapidly with any primary amine-containing molecule
• Membrane permeable, allowing for intracellular crosslinking
• High-purity, crystalline reagent can be used to create high-purity conjugates
• MS-cleavable
• Water-insoluble (dissolve first in DMF or DMSO)

Lot specifications:
• Purity: > 90% by quantitative NMR (the highest standard in crosslinking purity)

Chemical crosslinking in combination with mass spectrometry is a powerful method to determine protein-protein interactions. This method has been applied to recombinant and native protein complexes, and more recently, to whole cell lysates or intact unicellular organisms in efforts to identify protein-protein interactions on a global scale. Both traditional non-cleavable and MS-cleavable crosslinkers provide insight into the identification of protein-protein interaction sites, but MS-cleavable crosslinkers are advantageous due to their ability to be cleaved using different types of gas phase fragmentation methods (e.g., CID, HCD, ETD, and EtHCD) and levels of tandem mass spectrometry (e.g., MS2 and MS3) in order to improve identification of cross-linked peptides.

For high resolution analysis of the crosslinked peptides, the recommended LC column for Nanospray Flex source is Acclaim PepMap 100 C18 LC Column (Cat. No. 164940 or 164568). For EASY-Spray source, the recommended LC column is EASY-Spray C18 LC Column (Cat. No. ES800 or ES804).

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

The Thermo Scientific SureQuant RAS Isoform Panel (Absolute Quantitation) provides a complete reagent and quantitation solution for the enrichment and analysis of NRAS, HRAS, and KRAS isoforms using mass spectrometry (MS).

The SureQuant RAS Isoform Panel (Absolute Quantitation) consists of two modules:
SureQuant RAS Isoform IP and MS Sample Preparation Module includes all of the reagents necessary to immuno-enrich NRAS, KRAS, and HRAS proteins and perform in-solution MS sample preparation from sample lysis through peptide clean-up
SureQuant RAS 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 instrumentation and Nanospray LC instrumentation

SureQuant RAS 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 of RAS
Flexible—modular format allows for immuno-enrichment only, or in combination with relative or absolute quantitation

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 the 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 kit is ideal for monitoring expression levels of the three isoforms 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 RAS 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 RAS Isoform Absolute Quantitation Module provides reagents for absolute quantitation of three unique peptides from NRAS, KRAS, and HRAS 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 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

Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific™)

Thermo Scientific™ Pierce™ High pH Reversed-Phase Peptide Fractionation Kit increases protein identification from LC/MS analysis through orthogonal peptide fractionation of complex peptide samples.

Easy-to-use—resin provided in single-use spin column format
Improved protein identifications—protein identifications increased by ≥50% when compared to unfractionated samples
Reproducible—elution profiles and fractional resolution vary by less than 20%
Optimized—robust procedure for maximal protein identification and peptide recovery while minimizing fractional overlap
Compatible—reagents validated with a variety of complex samples, including TMT™-labeled peptides

In order to enable deep proteome sequencing, it is often necessary to reduce the sample complexity by fractionation in an orthogonal dimension prior to LC/MS analysis. The Pierce High pH Reversed-Phase Peptide Fractionation Kit utilizes high pH reversed-phase chromatography to separate peptides by hydrophobicity and provides excellent orthogonality to low pH reversed-phase LC-MS gradients.

The kit has been designed to improve protein identification through the use of a proprietary reversed-phase resin in an easy-to-use spin column format with a highpH fractionation protocol. In contrast to strong cation exchange (SCX) fractionation, the high-pH reversed-phase fractions do not require an additional desalting step before LC/MS analysis.

The High pH Reversed-phase Peptide Fractionation Kit includes a high pH buffer (0.1% triethylamine) and twelve spin columns containing pH-resistant reversed-phase resin. Each reversed-phase fractionation spin column enables fractionation of 10–100 µg of peptide sample using a microcentrifuge.

Native, phosphorylated, Tandem Mass TagTM (TMTTM)-labeled, and other complex peptide mixture samples can be fractionated using this kit. Combining the search results generated by the individual fractions helps improve protein sequence coverage and increases the number of identified proteins relative to unfractionated samples.

Applications:
• Reducing sample complexity to identify targets of interest
• Conducting systems biology studies
• Reducing sample complexity to improve quantitation studies

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

Thermo Scientific Glu-C Protease, MS Grade, is a highly specific endoproteinase use to improve sequence coverage in mass spectrometry protein identification applications.

Features of Glu-C Protease, MS Grade:

• Complementary to tryptic digests—hydrolyzes proteins specifically at the carboxyl side of glutamic acids
• Increased sequence coverage—better protein characterization results from overlapping peptides with complementary sequence coverage and chromatographic, ionization and fragmentation properties
• High specific activity—greater than 500 units/mg protein
• N-terminal arginine cleavage specificity—at least 90% for a complex protein sample
• Stable—provided in a lyophilized format

Product Details
This Glu-C is a mass spectrometry (MS)-grade serine protease isolated from Staphylococcus aureus. Glu-C can be used alone or alongside trypsin or other proteases to produce complementary protein digests for peptide mapping and protein sequencing. Glu-C protease is suitable for either in-solution or in-gel digestion workflows. This Glu-C enzyme is packaged lyophilized (5 x 10 µg).

The endoproteinase Glu-C, also referred to as V-8 Protease, specifically cleaves the carboxyl side of glutamic residues when reactions are carried out in ammonium bicarbonate and ammonium acetate buffers, generating a limited number of peptide fragments. Cleavage can also occur at both glutamic and aspartic residues in phosphate buffers. Glu-C can efficiently digest protein in 5-18 hours at 37°C. Glu-C Protease remains active under denaturing conditions such as 2M urea, 1M guanidine·HCl, 0.1% SDS, 2% CHAPS and 20% acetonitrile. Glu-C activity is optimal at pH 8. This lyophilized enzyme has a mass of 27 kDa and is stable for 1 year when stored at -20°C.

Applications
• Improved sequence coverage of protein digests
• In-solution digestion of proteins
• In-gel 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.

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

Pierce™ Trypsin Protease, MS Grade (Thermo Scientific™)

Thermo Scientific Pierce Trypsin Protease, MS Grade, is highly purified porcine trypsin that has been chemically modified for maximum activity and stability in proteomic applications.

Pierce Trypsin Protease, MS Grade, is a serine protease derived from porcine pancreatic extracts. The enzyme is TPCK-treated to eliminate chymotryptic activity and methylated to improve stability during protein digestion.

Features of Trypsin Protease, MS Grade:

Exceptional selectivity—cleaves at the carboxyl side of lysine and arginine residues with greater than 95% specificity
High purity—no detectable chymotrypsin activity
Enhanced stability—chemically modified for reduced autolytic activity

Applications:
• In-gel digestion of proteins from 1-D or 2-D gels
• In-solution tryptic digestion of proteins

Trypsin is a serine protease that specifically cleaves at the carboxyl side of lysine and arginine residues. The selectivity of this enzyme is critical for reproducible protein digestion and mass spectrometry-based protein identification. Since chymotrypsin co-purifies with trypsin derived from natural sources, Pierce Trypsin Protease has been treated with TPCK to eliminate chymotryptic activity, improving digestion specificity. Native trypsin is also subject to autolysis which can reduce enzyme stability and efficiency. To reduce autolytic degradation, Pierce Trypsin Protease is chemically modified by methylation, yielding a highly active and more stable form of the enzyme.

In addition to possessing high specific activity and being resistant to autolytic digestion, Pierce Trypsin Protease can tolerate commonly used partially denaturing conditions, such as 0.1% SDS, 1M urea and 10% acetonitrile. Pierce Trypsin Protease is most active in pH ranges pH 7 to 9 and can be reversibly inactivated at pH < 4. Both the liquid and lyophilized forms of the enzyme are stable for > 1 yearr when stored at -20°C.

Related Products
Pierce™ Trypsin Protease, MS Grade, Frozen