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

Thermo Scientific Pierce Trypsin/Lys-C Protease Mix, MS-Grade, is a mass spectrometry (MS)-grade serine endoproteinase mixture of trypsin and LysC that can be used for concurrent digestion of proteins for more efficient digestion than with trypsin alone.

Features of Pierce Trypsin/Lys-C Protease Mix, MS-Grade, include:
Enhanced digestion—enzyme combination reduces tryptic missed cleavages
Convenient—trypsin and LysC proteases provided at an optimized ratio for digestion in a combined-use format
Exceptional selectivity—trypsin has >95% C-terminal lysine and arginine specificity; LysC has >90% C-terminal lysine cleavage specificity
Stable—enzyme mixture provided in a lyophilized format

Protein characterization, identification, and quantification by MS begins with efficient, reproducible protein digestion. Although trypsin is routinely used for protein digestion, this protease alone is not sufficient to fully digest proteins at the carboxyl-end of lysine and arginine residues. Therefore, Lys-C protease is commonly combined with trypsin to sequentially digest proteins with fewer missed cleavages. Pierce Trypsin/Lys-C Protease Mix is a lyophilized mixture of trypsin and LysC proteases that has been optimized to improve digestion efficiency of proteins. It is provided in flexible formats of 20 µg, 5 x 20 µg, or 100 µg, and is also included in the EasyPep Mini MS Sample Prep Kit.

The MS-grade trypsin protease in this mix is derived from porcine pancreatic extracts and has been TPCK-treated to eliminate chymotryptic activity and methylated to improve stability during digestion. The MS-grade Lys-C protease is a highly purified native enzyme from <Lysobacter enzymogenes. Unlike trypsin, Lys-C can cleave lysines followed by prolines, making it ideal for use in combination with other proteases for optimal protein digestion. When used as a mixture, digestion can be completed in as little as 1.5–3 hours or up to overnight, depending on enzyme to protein ratio. This lyophilized enzyme mixture is stable for one year when stored at -20°C.

Related products
EasyPep Mini MS Sample Prep Kit
Pierce Colorimetric Peptide Quantitation Assay
Chloroacetamide, No-Weigh Format

Pierce™ Chymotrypsin Protease (TLCK treated), MS Grade (Thermo Scientific™)

Thermo Scientific Chymotrypsin Protease, MS Grade, is a purified native endoproteinase validated for maximum activity and selectivity in proteomic applications.

Features of Chymotrypsin Protease, MS Grade:

• Increased sequence coverage—cleaves at the carboxyl side of tyrosine, phenylalanine, tryptophan and leucine
• Better specificity—treated with TLCK to eliminate trypsin activity
• High activity—greater than 45 units/mg protein

This Chymotrypsin is a mass spectrometry (MS)-grade serine protease isolated from bovine pancreatic extracts. This chymotrypsin is TLCK-treated to eliminate tryptic activity. The selectivity of this enzyme is important for reproducible protein digestion and mass spectrometry-based protein identification. Chymotrypsin-digestion typically generates a larger number of shorter peptides compared to trypsin. This mass spectrometry-grade Chymotrypsin is packaged lyophilized (convenient 4 x 25 µg).

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

Product Details
The endoproteinase chymotrypsin specifically cleaves at the carboxyl side of tyrosine, phenylalanine, tryptophan and leucine. Two predominant forms of chymotrypsin, A and B, are found in equal amounts in bovine pancreas. They are similar proteins (80% homology), but have different proteolytic characteristics. Both forms of chymotrypsin are present in Thermo Scientific Chymotrypsin. Since trypsin may co-purify with Chymotrypsin derived from natural sources, Thermo Scientific Chymotrypsin has been treated with TLCK to eliminate potential tryptic activity, improving digestion specificity. Chymotrypsin can tolerate mild denaturing conditions, such as 0.1% SDS, 2M urea, 2M guanidine·HCl, 1% CHAPS, and 30% acetonitrile with optimal activity in the pH range 7.5 to 8.5. This lyophilized enzyme has a mass of 25 kDa and is stable for 1 year when stored at -20°C.

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

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

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.

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

In-Gel Tryptic Digestion Kit (Thermo Scientific™)

Thermo Scientific Pierce In-Gel Tryptic Digestion Kit provides a complete set of reagents for digesting and recovering peptide fragments from approximately 150 stained protein bands excised from SDS-PAGE (polyacrylamide gel plugs).

Features of the kit:

Convenient—includes all necessary reagents for destaining coomassie- or fluorescent dye-stained proteins, reduction and alkylation of cystines, and tryptic digestion
Robust—the procedure and reagents produce reliable digestions and data generation using a wide range of conditions without optimization
Accurate—contains highly purified and modified MS-grade trypsin that shows no chymotryptic activity and minimal autolytic activity

The In-Gel Tryptic Digestion Kit provides a complete set of reagents for performing approximately 150 digestions on colloidal coomassie- or fluorescent dye-stained protein bands. The kit includes modified porcine trypsin, destaining buffers, reduction reagents, alkylation reagents and digestion buffers along with detailed and simple instructions. Each component and step has been optimized and balanced to produce complete, accurate and clean digests using a variety of conditions for dependable proteomics analysis of peptide profiles by mass spectrometry (MS).

Accurate identification and analysis of proteins is a central component of proteomic strategies for studying cellular functions and processes. Mass spectrometers are essential tools for these studies as they provide a high level of sensitivity and mass accuracy. However, this sensitivity and accuracy can be obtained only if proper sample preparation is performed. Two key sample preparation steps are the fragmentation of proteins into peptides and subsequent concentration and clean up of peptides. The In-Gel Tryptic Digestion Kit accomplishes the first of these steps. Thermo Scientific Pierce C-18 Columns accomplish the second step.

SureQuant™ TP53 Panel IP and MS Sample Preparation Module (Thermo Scientific™)

The Thermo Scientific SureQuant TP53 Panel IP and MS Sample Preparation Module provides a complete reagent solution for the multiplexed enrichment and sample preparation of TP53 for mass spectrometry (MS).

SureQuant TP53 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 TP53 and phosphorylated TP53 protein

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 module is ideal for multiplexed immuno-enrichment of TP53 protein for use with the SureQuant quantitation modules.

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 and 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 TP53 Panel Absolute Quantitation Module or the SureQuant TP53 Panel 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™ LC-MS/MS System Suitability Standard (7 x 5 Mix) (0.5 pmol/µL) (Thermo Scientific™)

The Pierce LC-MS/MS System Suitability Standard (7 x 5 mixture) contains seven HeavyPeptide AQUA Ultimate peptides provided at five dilutions distinguished by differential isotopic labeling to assess sensitivity and dynamic range of LC-MS/MS systems. The standard contains 7 of 15 Pierce Retention Time Calibration Mixture (Cat. No. 88320) peptides. Each of the seven peptides has five isotopologue versions containing no heavy, 1 heavy, 2 heavy, 3 heavy, and 4 heavy isotope-labeled amino acids. The isotopologues for each peptide are provided at a distinct concentrations to generate seven standard curves to assess system performance. The 7 x 5 mixture can be used with Skyline software (www.thermoscientific.com/pinpoint) to assess dynamic range, linearity, and lower limit of quantitation (LLOQ).

Pierce LC-MS/MS System Suitability Standard (7 x 5 mixture) features include:
High quality—contains five sets of seven HeavyPeptide AQUA Ultimate peptides, with each peptide distinguishable through unique isotopologue labeling
Optimized—enables scheduling of MS acquisition windows for improved quantification and increased multiplexing
Quantitative—enables assessment of dynamic range of instrument and instrument sensitivity
Predictive—enables assessment of chromatography and MS instrument performance

Applications:
• Standard for data-dependent acquisition (DDA)
• Standard for targeted MS (e.g., parallel-reaction monitoring (PRM))
• Optimization of LC parameters
• Identification of total peptide elution window
• Optimization of MS parameters
• Assessment dynamic range of nano or capillary flow LC-MS/MS systems

Related products:
EASY-Spray LC Analytical Column
Acclaim PepMap 100 C18 Trap Column, NanoViper
Pierce TMT11plex Yeast Digest Standard

General reference:
1. Krokhin OV, Spicer V. Peptide retention standards and hydrophobicity indexes in reversed-phase high-performance liquid chromatography of peptides. Anal Chem. 2009 81(22):9522-30.

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

Pierce™ Peptide Desalting Spin Columns (Thermo Scientific™)

Thermo Scientific Pierce Peptide Desalting Spin Columns are ready-to-use centrifuge spin columns that enable efficient desalting of peptide samples following enzymatic digestion. The polymer-based hydrophobic resin contained in each column provides excellent binding and recovery characteristics for peptide samples in preparation for mass spectrometry and other methods.

Features of Pierce Peptide Desalting Spin Columns include:
Removes MS-interfering contaminants—significantly reduces signal suppression and improves sensitivity in mass spectrometry
High binding capacity—special hydrophobic polymer-based resin allows for excellent recovery of peptide loads of 5 µg to as high as 5 mg of native and TMT-labeled peptides
Easy to use—resin is provided in single-use spin column format that fits many common 2 mL tubes
Compatible—resin is validated with a variety of complex samples, including TMT-labeled peptides, and is stable under extreme pH conditions

Pierce Peptide Desalting Spin Columns provide a convenient and reproducible way to desalt and remove contaminants from peptide samples prior to analysis by mass spectrometry. Each spin column can bind from 5 µg to 5 mg of native and TMT-labeled peptides. The spin column format allows processing of multiple samples (10 to 300 µL each) in parallel in approximately 30 minutes.

Mass spectrometry is essential for studying biological compounds. However, many of the buffers and reagents used in the course of sample preparation will interfere with mass spectrometry analysis, leading to poor-quality spectra and reduced sensitivity. Pierce Peptide Desalting Spin Columns are designed to remove many of the interfering hydrophilic sample contaminants with minimal sample loss, thus enabling sensitive and comprehensive analysis of digested protein samples by mass spectrometry.

High Select™ HSA/Immunoglobulin Depletion Mini Spin Columns (Thermo Scientific™)

Thermo Scientific High Select HSA/Immunoglobulin Depletion Resin decreases the albumin and antibody components that are abundant in human plasma samples, helping prepare those samples for mass spectrometry or 1D or 2D electrophoresis. The resin is provided here in a convenient single-use mini spin column format for the treatment of 10 µL samples.

Features of HSA/IgG Depletion Resin include:
• Flexible—mini column format is scaled and optimized for treating 10 µL human plasma samples
• Optimized—removes >95% of IgG and >95% of albumin
• Fast—process samples in ~10 minutes
• Economical—cost-effective spin columns are priced for single use
• Reduce variability—one-time use prevents protein carryover and experimental variability

High Select HSA/Immunoglobulin Depletion Resin uses highly specific immobilized anti-HSA and anti-Immuoglobulin antibodies (IgG, IgA, IgM, IgD, and IgE) to remove human serum albumin (HSA) and all major subclasses of immunoglobulins from serum and plasma. The resin is provided in an economical and convenient spin column format specifically designed for one-step processing and single use.

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 HSA/Immunoglobulin Depletion Resin can deplete greater than 95% of HSA and 95% of IgG. Samples are loaded in a pre-filled disposable spin column and processed in approximately 10 minutes. The depletion of highly abundant proteins helps enable the detection of low abundant proteins in samples and subsequent identification by mass spectrometry or 1D or 2D gel electrophoresis.

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TMT10plex Isobaric Label Reagent Set, 3 x 0.8 mg

NeuCode™ Lysine-080 (Thermo Scientific™)

Thermo Scientific™ NeuCode™ amino acids augment the level of multiplexing achievable for the metabolic labeling of proteins for mass spectrometry analysis. NeuCode™ Lysine-080 (3,3,4,4,5,5,6,6-D8 L-Lysine-2HCl) may also be used with traditional SILAC to improve flexibility of multiplexing options or to reduce complexity of analysis.

General features of NeuCode SILAC labeling:
• Labeling efficiency—100% label incorporation into proteins of living cells without toxicity
• Compatible—may be multiplexed with existing SILAC amino acids
• Time-saving—not necessary to label to 100% incorporation if only using heavy amino acids
• High-quality supplements—heavy amino acids with >98% isotope purity

Stable isotope labeling with amino acids in cell culture (SILAC) is a powerful method to identify and quantify relative differential changes in complex protein samples. NeuCode metabolic labeling is similar to SILAC, but differs in that the labeling only utilizes heavy amino acids. The increased multiplexing capability of NeuCode amino acids is possible through the use of mass defects from extra neutrons in the stable isotopes. These small mass differences may be resolved on high resolution mass spectrometers (Thermo Scientific™ Orbitrap™ Elite™, Q Exactive™, Orbitrap™ Fusion™ Tribrid™, and Orbitrap™ Fusion™ Lumos™ Tribrid™ mass spectrometers). Use of only heavy amino acids eliminates the need for 100% incorporation of amino acids used for SILAC (both heavy and light), and may be especially useful for studies with primary cells.

NeuCode amino acids are used together with specialized cell culture media that are deficient in essential amino acids. Heavy L-lysine is used for SILAC analysis of peptides that have been digested with trypsin or LysC. NeuCode Lysine-080 may be used with 4,4,5,5-D4 L-lysine or 13C6 15N2 L-lysine for duplex experiments and may be combined with light lysine for three-plex experiments.

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L-Lysine-2HCl, 4,4,5,5-D4 for SILAC
L-Lysine-2HCl, 13C6, 15N2 for SILAC
Fetal Bovine Serum, dialyzed, US origin
DMEM Media for SILAC

NeuCode is a trademark of WARF.

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

The Thermo Scientific SureQuant RAS Isoform Panel (Relative 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 (Relative 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 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 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 Relative Quantitation Module provides reagents for relative quantitation of three unique peptides from NRAS, KRAS, and HRAS 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

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