Thursday, 28 June 2018

Proteomics -Targeted proteomics

Biognosys’ label-free targeted proteomic workflows allow absolute quantification of up to 150 pre-defined proteins that can be analyzed in a high-throughput mode in thousands of samples. In contrast to HRM where all peptides and fragment ions are recorded, targeted proteomics limits the number of peptides that will be monitored and only focuses on those peptides during acquisition to achieve the highest sensitivity and throughput for hundreds or thousands of samples. Targeted proteomics allows absolute quantification of the proteins of interest by including stable isotope standards in the analysis.
Targeted proteomics currently relies on two main approaches: Multiple and Parallel Reaction Monitoring (MRM and PRM). MRM is a well-established method for targeted proteomics and is primarily performed on triple quadrupole mass spectrometers, while its novel variant PRM was introduced recently and is performed on the latest generation of high-resolution mass spectrometric instruments.

Targeted proteomics is based on three essential parts:

Assay development and set-up: This is the first step in targeted proteomics workflows and resembles spectral library generation in HRM. For the proteins of interest a collection of assays (also called transition or exclusion lists) is built and optimized. An assay describes the characteristic features of peptide spectra such as fragment ion intensities and retention time (iRT). These assays are later used in data acquisition.
Data acquisition: Data acquisition is performed on triple quadrupole (MRM) or high-resolution mass spectrometric instruments (PRM). In both approaches, an upfront quadrupole is used to isolate the targeted precursor ions from the assay list followed by a collision cell where product ions (transitions) are generated. While MRM only measures pre-selected transitions with the third quadrupole, PRM detects all product ions in a high-resolution mass analyzer increasing the number of quantifiable proteins in one run.
Data analysis is historically a limitation in high-throughput analysis of multiplexed MRM and PRM runs while a significant manual input is required with available tools. Biognosys overcomes these challenges with its proprietary software SpectroDive that features smartest peak picking algorithms and enables extremely fast analysis of large datasets.
Biognosys’ targeted proteomics platform is most suitable for verification or validation studies when many samples have to be analyzed for a pre-determined set of proteins of interest.

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