SureQuant Targeted Quantitation

A new paradigm for targeted protein quantitation

In combination with Thermo Scientific SureQuant targeted assay kits and LC-MS Orbitrap MS, the SureQuant IS targeted quantitation workflow delivers a complete mass spectrometry assay from sample preparation to monitoring and quantitation of target peptides, with easy set up and high confidence in results.

The high selectivity of HRAM Orbitrap MS has enabled a new paradigm for targeted proteomics, delivering a highly sensitive approach able to quantify hundreds of protein targets in complex matrices with high quantitative accuracy, precision and specificity.

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Thermo Scientific SureQuant Targeted Mass Spec Assay kits

More targets with no compromise in sensitivity

Thermo Scientific SureQuant Targeted Mass Spec Assay kits are validated, modular reagents for multiplexed target protein quantitation. These complete kits contain all the reagents needed for successful monitoring of MS performance, sample preparation and absolute quantitation of target proteins/peptides. Verified kits means that all antibodies, peptides and control lysates are rigorously tested for specificity and successful quantitation of each target peptide. Each SureQuant kit is also fully validated and optimized for use with the SureQuant instrument method templates so all of the assay development time is completely removed. Easy to use, validated Skyline data processing templates are also provided with each kit to ensure you go from assay to insight in no time.

Orbitrap Exploris 480 Mass Spectrometer

Making genius simpler

Orbitrap Exploris 480 Mass Spectrometer is the latest instrument which delivers proven technology, advanced capabilities, a compact footprint and an intelligence-driven analytical framework empowering research scientists to more extensively deploy mass spectrometry for rigorous, high-throughput protein identification and quantification. The Orbitrap Exploris MS works with the SureQuant quantitation workflow to provide a data-aware quantitation scan mode which leverages internal standards to dynamically adjust scan parameters and automatically maximize data quality for targeted proteome analysis in real time.

Orbitrap Eclipse Tribrid Mass Spectrometer

One system, maximum insights to go beyond today’s discovery

Orbitrap Eclipse Tribrid Mass Spectrometer surpasses typical LC-MS limits with new innovations that deliver the ultimate flexibility to expand experimental scope, with built-in intelligence to ensure the highest data quality and confidence. One system provides maximum insights, so more users can productively go beyond today’s discovery. With the SureQuant quantitation workflow it reduces the inevitable trade-off between target multiplexing, sensitivity and selectivity and enlarges targeted protein panel sizes, significantly improving limits of detection (LODs) and quantitative accuracy without use of retention time windows.

Skyline Software

Skyline software  is widely adopted by the proteomics community as the de facto platform for targeted quantitation data analysis. The SureQuant Targeted Mass Spec Assay kits are provided with ready-to-use Skyline processing and analysis templates to enable routine workflow execution without tedious parameter and data pipeline optimization.

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How the SureQuant workflow overcomes traditional limitations

Most biochemical functions of the cell are performed by proteins, and they’re also the target of most current drugs. To understand the functions of proteins and their role in complex biological systems, it’s necessary to measure changes in protein abundance relative to those of the system. The advent of highly sensitive mass spectrometry techniques has enabled relative quantitation experiments of proteins at a proteome-wide scale between samples and biological systems.

But, some fundamental challenges remain in measuring proteins:

  • Dynamic range of expression can vary in different matrix types, 6-7 orders of magnitude in tissues, and approximately 12 orders of magnitude in body fluids such as plasma and serum
  • Proteoforms must be considered; these arise from alternative splicing of pre-RNA
  • Post translational modifications (PTMs) add to the variation of proteoforms

Once proteins have been identified as changing in a relative quantitation experiment or from hypothesis driven strategies, these proteins need to be validated and verified across a larger sample population. The measurements of these target proteins can be relative or absolute levels; this approach is called targeted quantitation.

The challenge of validation with traditional targeted protein quantitation

Traditional targeted protein quantitation assays are based on affinity reagent based methods such as ELISAs, western blotting or immunohistochemistry staining. These techniques are limited by dynamic range, specificity, sensitivity and the small number of targets that are detectable and quantifiable.

Targeted protein quantitation with mass spectrometry delivers reliable, large-quantity results

Targeted protein quantitation using mass spectrometry is best suited to overcome these limitations. Traditionally, selected reaction monitoring (SRM)-MS on a triple quadrupole mass spectrometer has been the gold standard for targeted experiments, as long as protein targets are not in complex matrices, and the number of targets are small (20-100 peptides).

The SureQuant IS targeted protein quantitation workflow with HRAM Orbitrap MS delivers sensitive multiplexed quantitation of a large number of targets with unrivaled confidence. It enables life scientists to quantify proteins using the unique, intelligent, real-time monitoring of targets with highly sensitive mass spectrometry. By leveraging labeled internal standard peptides to guide the acquisition of endogenous peptides in real time, reliable quantitation of hundreds of targets becomes a reality without compromise and with easy method set-up.

The overall SureQuant IS targeted protein quantitation workflow is comprised of two steps:

  1. Survey Run to verify the detectability of the reference internal standards (IS);
  2. SureQuant Method to monitor expression and quantification of endogenous/target peptides.

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Step 1: Survey Run to determine intensity of internal standards

The Survey Run analysis evaluates the detection of the internal standards supplemented into a representative sample matrix. It only needs to be run one time at the onset of the study, on a user’s preferred LC-MS configuration, and no further adjustment is required over time. This analysis verifies the optimal precursor ion of each IS peptide and the optimal associated fragment ions can be detected. The signal intensity of the IS and subsequent triggering intensity is also determined from the Survey Run. Data processing is supported by Skyline, enabling the export of these pieces of information, which can be transferred then to the SureQuant method through simple copy/paste. The required information is imported into the SureQuant method and then after this one-time adjustment the method is ready for routine, targeted analysis of samples with spikes in IS.

SureQuant Survey Run determines the intensity of internal standards
 Click to enlarge

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Step 2: SureQuant Method to monitor, detect and quantify

The Thermo Scientific SureQuant Method is an MS acquisition strategy where the mass spectrometer is programmed to monitor reference IS in the sample using low fill times and resolution and dynamically increase these parameters, upon their detection, to enhance the data quality for endogenous peptides. The real-time management of acquisition time maximizes the time devoted to analyte quantitation allowing greater number of targets to be reliably detected and quantified for targeted proteomics experiments. The built-in positive IS control provides a definitive LOD measure for the presence or absence of proteins in the sample, addressing the common need to assess protein copy number expression in many molecular biology experiments.

SureQuant Method enables greater peptide detection and quantification
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Save time with built-in SureQuant validated instrument method templates

Validated instrument method templates for both Survey Run and SureQuant Method analysis are provided, preset for various SureQuant Targeted MS assay kits like the AKT/mTOR pathway kit. In these templates, peptide precursors, reference fragment ions, and instrument parameters are optimized and preset—meaning zero assay development time. Simply spike in internal standards and collect data. Generic SureQuant Method templates are also available to simplify the development of custom protein panel assays.

Kit specific and generic SureQuant validated instrument method templates
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Products for the complete SureQuant IS targeted protein quantitation workflow

Each step of the workflow below is optimized for simplicity and reproducibility, targeting hundreds of targets in a single sample with the highest quantitative accuracy and precision.

Steps in the SureQuant targeted protein quantitation workflow
 Click to enlarge
Thermo Scientific SureQuant Targeted MS Assay Kits

Multiplex immunoprecipitation to mass spectrometry (mIP-MS) for the simultaneous enrichment and quantitation of target proteins. Each multiplex panel contains two modules: 1) an IP and MS sample prep module which includes all the reagents necessary to immunoenrich AKT/mTOR pathway proteins and perform in-solution MS sample preparation, and 2) an absolute or relative quantitation module, which includes a system suitability standard and AQUA Ultimate Heavy and/or Light Peptide mixtures.

EasyPep Mini MS Sample Prep Kit

Prepare your samples in less than 3 hours using pre-formulated reagents for analysis by mass spectrometry.

HeavyPeptide AQUA Custom Synthesis Service

Isotopically labeled, AQUA-grade peptides for the relative and absolute quantitation of proteins at very low concentrations in complex protein mixtures.

Acclaim PepMap 100 C18 LC Columns

Obtain high resolution separations of low abundant peptides in complex proteomic samples using columns with high loading capacity.

Trap Column Cartridges Holders with nanoViper Fittings

Thermo Scientific Trap Column Cartridges Holders with nanoViper fittings do not "grab" the tubing at a single point but seal at the tip without using a ferrule. This gives nanoViper fittings their UHPLC, ensuing virtually dead-volume-free capabilities.

EASY-Spray LC Columns

Ensure robust nano and capillary flow LC-MS with Thermo Scientific EASY-Spray LC Columns. The integrated column/emitter design eliminates dead volume and is temperature-controlled for maximum reliability and performance. Rigourously tested to ensure maximum quality, these columns are designed for maximum simplicity and ease-of-use.

EASY-nLC 1200 System

Experience performance and throughput at 1200 bar with the easy to operate nano-flow UHPLC instrument.

UltiMate 3000 RSLCnano System

Take advantage of the wide flow rate range which gives you the flexibility you need for low flow applications.

Orbitrap Exploris 480 Mass Spectrometer

Delivers best in class SureQuant IS targeted protein quantitation for hundreds of targets. By leveraging intelligent acquisition for real time monitoring of targets, proteins are quantified with the highest confidence with easy method setup within a remarkably compact footprint.

Orbitrap Eclipse Tribrid Mass Spectrometer

Delivers the ultimate flexibility to expand experimental scope, and with built-in intelligence ensures the highest data quality and quantitation for several quantitative proteomics workflows including SureQuant IS targeted quantitation.

Skyline software

Skyline software  is widely adopted by the proteomics community as the de facto platform for targeted quantitation data analysis. The SureQuant Targeted Mass Spec Assay kits are provided with ready-to-use Skyline processing and analysis templates to enable routine workflow execution without tedious parameter and data pipeline optimization.

Thermo Scientific SureQuant Targeted MS Assay Kits

Multiplex immunoprecipitation to mass spectrometry (mIP-MS) for the simultaneous enrichment and quantitation of target proteins. Each multiplex panel contains two modules: 1) an IP and MS sample prep module which includes all the reagents necessary to immunoenrich AKT/mTOR pathway proteins and perform in-solution MS sample preparation, and 2) an absolute or relative quantitation module, which includes a system suitability standard and AQUA Ultimate Heavy and/or Light Peptide mixtures.

EasyPep Mini MS Sample Prep Kit

Prepare your samples in less than 3 hours using pre-formulated reagents for analysis by mass spectrometry.

HeavyPeptide AQUA Custom Synthesis Service

Isotopically labeled, AQUA-grade peptides for the relative and absolute quantitation of proteins at very low concentrations in complex protein mixtures.

Acclaim PepMap 100 C18 LC Columns

Obtain high resolution separations of low abundant peptides in complex proteomic samples using columns with high loading capacity.

Trap Column Cartridges Holders with nanoViper Fittings

Thermo Scientific Trap Column Cartridges Holders with nanoViper fittings do not "grab" the tubing at a single point but seal at the tip without using a ferrule. This gives nanoViper fittings their UHPLC, ensuing virtually dead-volume-free capabilities.

EASY-Spray LC Columns

Ensure robust nano and capillary flow LC-MS with Thermo Scientific EASY-Spray LC Columns. The integrated column/emitter design eliminates dead volume and is temperature-controlled for maximum reliability and performance. Rigourously tested to ensure maximum quality, these columns are designed for maximum simplicity and ease-of-use.

EASY-nLC 1200 System

Experience performance and throughput at 1200 bar with the easy to operate nano-flow UHPLC instrument.

UltiMate 3000 RSLCnano System

Take advantage of the wide flow rate range which gives you the flexibility you need for low flow applications.

Orbitrap Exploris 480 Mass Spectrometer

Delivers best in class SureQuant IS targeted protein quantitation for hundreds of targets. By leveraging intelligent acquisition for real time monitoring of targets, proteins are quantified with the highest confidence with easy method setup within a remarkably compact footprint.

Orbitrap Eclipse Tribrid Mass Spectrometer

Delivers the ultimate flexibility to expand experimental scope, and with built-in intelligence ensures the highest data quality and quantitation for several quantitative proteomics workflows including SureQuant IS targeted quantitation.

Skyline software

Skyline software  is widely adopted by the proteomics community as the de facto platform for targeted quantitation data analysis. The SureQuant Targeted Mass Spec Assay kits are provided with ready-to-use Skyline processing and analysis templates to enable routine workflow execution without tedious parameter and data pipeline optimization.

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Featured scientific posters on the SureQuant quantitation method