SureQuant Targeted Quantitation

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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• Featured products for SureQuant targeted quantitation
• How the SureQuant workflow overcomes traditional limitations
• Step 1: Survey Run to determine intensity of internal standards
• Step 2: SureQuant Method to monitor, detect and quantify
• Save time with built-in SureQuant validated instrument method templates
• Products for the complete SureQuant IS targeted protein quantitation workflow
• Featured scientific posters on the SureQuant quantitation method
• Resources and support

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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
   

Complexity of the proteome

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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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 and Proteome Discoverer 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.

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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.

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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.

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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.

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