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Targeted Protein Quantitation - The next step in the search for biomarkers
A common endpoint for a biomarker discovery experiment is a list of putative marker proteins. A reasonable next step is to perform targeted quantitative measurements of these proteins in an expanded patient population to verify their statistical significance. Unlike in the discovery phase, hundreds of samples need to be run for statistical verification. A robust, high-throughput method that offers both specificity and sensitivity is required.
The SRM approach
The most common analytical approach to this challenge is to use selected-reaction monitoring (SRM) on a triple quadrupole mass spectrometer to monitor a unique peptide or group of unique peptides for each protein of interest. SRM on a triple quad provides an outstanding combination of specificity, sensitivity, and reliability, making it ideal for this application. Isobaric mass tags that enable concurrent identification and quantitation of proteins in different samples are sometimes incorporated into this workflow.
The high-resolution advantage
Although SRM by triple quadrupole MS gives very specific and sensitive responses for targeted peptides, the resolution limit of the first quadrupole (Q1) can make it difficult to differentiate between a targeted precursor and chemical background or a non-targeted peptide, particularly when detecting very-low-abundance peptides in highly complex samples. It can also prevent the use of valuable precursor-to-product transitions if other precursor peptides in the sample have similar masses and product ions.
The Thermo Scientific TSQ Quantum Ultra and TSQ Vantage triple quadrupole mass spectrometers provide an effective solution to this challenge. Whereas most triple quads are limited to unit mass resolution (0.7 Da FWHM) for precursor selection, the TSQ Quantum Ultra™ and TSQ Vantage™ can employ high-resolution precursor selection (0.2 Da FWHM). The additional specificity of this high-resolution selected reaction monitoring (H-SRM) reduces interferences, providing more accurate quantitation and more robust assays without loss of signal. Compared to competing instruments, it reduces the need to discard transitions that would otherwise provide additional specificity.
- H-SRM dramatically reduce interferences from complex backgrounds, improving overall assay specificity
- H-SRM reduces the need to discard useful transitions due to similar transitions in other sample peptides
- Thanks to fast H-SRM cycle times, more transitions can be monitored for increased specificity and confidence
- H-SRM increases confidence in verification strategies such as QED – SRM-triggered acquisition of full-scan MS/MS or pseudo MS/MS spectra
A seamless transition from discovery to targeted quantitation
Thermo Fisher LTQ- and LTQ Orbitrap-based discovery experiments provide a wealth of MS and MS/MS information that can be leveraged to build effective targeted quantitative assays. Thermo Scientific Pinpoint software enables a seamless transition from biomarker discovery to targeted protein quantitation. The software:
- Predicts proteotypic peptides
- Determines the best SRM transitions and builds the TSQ instrument method
- Quantitates each targeted peptide
- Utilizes MS/MS data from discovery experiments to optimize the quantitation method
Obtain more information and download the software here.
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