Recent Proteomics Webinar from Thermo Fisher Scientific

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There is an increased need to multiplex protein quantification for protein biomarker verification and to perform large-scale protein quantification for modeling systems biology. It is difficult to develop traditional immunoassays (ELISA) for multiplex protein quantification. Such assays can be expensive, and time and resource intensive. Alternatively, selected-reaction monitoring (SRM) by tandem mass spectrometry can be used for complex quantitative protein assays. This approach affords the selectivity of two stages of mass selection, the sensitivity of monitoring only selected ions, wide dynamic range, and high throughput.

One of the greatest challenges of developing SRM assays for large-scale protein quantification is selecting the optimal SRM transitions without access to the appropriate peptide standards. Thermo Scientific Pinpoint software provides a means to utilize libraries of MS/MS spectra from Thermo Scientific LTQ ion trap-based discovery experiments to speed the selection and optimization of SRM transitions. It dramatically reduces the difficulty of selecting "proteotypic" peptides and their transitions. The software features a user-friendly, workflow-driven interface to guide the user from the discovery phase to targeted protein quantitation experiments

Traditional multiple SRM experiments suffer from a compromise between high-sensitivity detection and rigorous peptide verification. The newly introduced intelligent SRM (iSRM) technique, uses instrument time very efficiently, providing simultaneous high-sensitivity quantification and verification of hundreds of peptides in a single HPLC MS run. The iSRM approach defines primary and secondary SRM transitions for each peptide. Primary SRM transitions are used to quantitate expression levels. They also trigger, in a data-dependent fashion, the acquisition of secondary SRM transitions resulting in a composite MS/MS spectrum for confident verification. This approach significantly increases duty cycle of the instrument, facilitating rapid development and application of complex SRM assays for large scale screening.

In this webinar, we demonstrate the complete targeted protein quantification workflow step-by-step: building SRM tables, data acquisition and processing methods, followed by automated results verification, and finally method refinement.

Your presenter

Scott Peterman
Strategic Marketing, Proteomics
Thermo Fisher Scientific