Transitioning from a discovery experiment intended to characterize what proteins are in a sample to obtaining the relative abundance of those proteins using highly sensitive targeted quantitation methods (e.g., PRM) can present a substantial obstacle to many research pipelines. Data Independent Analysis (DIA) bridges protein identification and targeted quantitation by acting as a screening technique to rapidly and thoroughly survey a sample and direct future targeted analysis. DIA creates a comprehensive record of a sample by sequentially scanning all ions across a wide mass-to-charge range ensuring that all detectable parent ions and fragments are examined. This technique is entirely untargeted; there is no scheduling of transitions and thus no unselected peptides, no mistimed transitions. Nevertheless, not all DIA methods are created equal: Speed, sensitivity, and selectivity form an ideal limit on how much information can be extracted from a DIA experiment.
In this webinar, we will cover building high performance methods on the Orbitrap Fusion Tribrid MS and Q Exactive HF MS to take advantage of analyzer parallelization with WiSIM and precursor multiplexing with msxDIA. You will learn how to generate a high quality spectral library and use it to reveal important expression changes in your pathway of interest, and even how to go back in time and use a DIA file’s record observable proteome to retrospectively interrogate data for new target peptides and proteins. Finally we will demonstrate how DIA can be used to select appropriate targets for further more sensitive SRM/PRM analysis and how reducing sample complexity with immunoprecipitation can significantly enhance the sensitivity of DIA allowing its use as a satisfactory alternative to targeted methods.
Key learning objectives
- What are the DIA methods and how they influence speed, sensitivity, and selectivity
- How to obtain comprehensive and reproducible characterization of a biological pathway
- Learn how to generate a high quality spectral library and use it to reveal important expression changes in your pathway of interest
- Learn how you can retrospectively interrogate data for new target peptides / proteins
Who should attend
- Protein and peptide biochemists
- Mass spectrometrists
- Core lab directors
Michael Blank obtained his doctorate in chemistry from Stanford University. Since joining Thermo Fisher Scientific, he has been involved in several projects focused on protein quantitation including isobaric tagging, label free quantitation, targeted assays, and data independent analysis. He has also been associated with several instrument launches including the Thermo ScientificTM Orbitrap FusionTM TribridTM MS and the Thermo ScientificTM Q ExactiveTM HF MS.