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Abstract Introduction
Targeted proteomics analysis based on Multiple Reaction Monitoring approach (MRM) allows high specificity, sensitivity and reproducibility in complex biological matrices, in regards to its intrinsic properties. This latter is thus considered as the golden approach for quantification purposes and a growing interest appeared, particularly in biomarker discovery field and clinical analysis. However, this analysis mode is currently limited by its multiplexing capabilities. According to scheduled MRM mode (sMRM), this limitation was over-passed, but sMRM assay development remains complicated and time consuming. Also, Retention Times (RTs) must be known and reproducible. This constraint is poorly compatible with routinely clinical analysis in which RTs shifts occur due to both instrumental system variations and samples differences induced by biological heterogeneity. A new approach, the so-called SCOUT-MRM approach was developed, which keeps sMRM multiplexing capabilities without RTs constraints.
Objectives
The following poster presents “peptide selector”, a new tool to automatically implement a SCOUT-MRM method. This tool, coupled to SCOUT-MRM-BEADS kit (based on READYBEADSTM technology) was developed to automatically rank peptides of interest in the appropriate SCOUT groups. SCOUT groups peptides monitoring is then triggered by detection of reporter peptides (SCOUT-MRM-BEADS kit), independently of RT. This approach opens new opportunities to develop highly multiplexed MRM assay, individually for each sample, with an easy straightforward fashion. Also, it ensures high reproducibility between samples, since all peptides are detected even with high RTs shifts between them. For clinical analyses, data completeness and robustness (detect and identify all peptides in each analysis) are a requirement, and this approach brings a serious analytical answer.
Method
To illustrate and highlight the interest of “peptide selector” software, coupled to SCOUT-MRM-BEADS kit for multiplex analysis, 58 peptides from 40 proteins were monitored in serum samples, either with classical sMRM method, or with automatically-generated SCOUT-MRM method.
Results
18 peptides were detected after screening a plasma sample with conventional sMRM method, while SCOUT-MRM automatically-generated method allowed detecting the 58 peptides.
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= Discovery stage.
= Translation stage.
= Clinically available.
