I kicked off the scientific presentations for the virtualized Northeastern University May Institute with a workshop on the basics of mass spectrometry proteomics. In this mixed-methods 1.5 hour session, I aimed to give biomedical researchers a crash course in all things quantitative mass spectrometry-based proteomics and even give some of the pros a few tips on the Skyline software ecosystem. By the end of this workshop, I wanted participants to come away with the ability to:
Assess the experimental pros and cons of targeted proteomics, and compare to discovery proteomics.
Explain the fundamentals of mass spectrometry proteomics, including peptide fragmentation and basic components of a mass spectrometer
Describe the steps of a targeted proteomics workflow and the information required to build an assay
Apply the Skyline software ecosystem to their own targeted proteomics experiments.
Unsurprisingly, my most common approach to proteome abundance measurements by mass spectrometry is data independent acquisition (DIA). Specifically I’ve been using the chromatogram library approach (Searle et al 2018) because, compared to spectral library-based approaches, it doesn’t take a lot of extra work. I just prepare my samples as usual, then pool a few uL of each sample into a “library” or consensus sample. I queue up my single-shot experimental samples, then I acquire the pooled library sample with multiple injections, each time spanning a 100 m/z range (gas phase fractionation, GPF) with very narrow isolation windows.
The next step up is to search the narrow window, GPF multi-injections against a spectral library. Recently, a team of researchers released “Prosit”, a tool to predict spectral libraries. Using Prosit predicted spectral libraries to search GPF chromatogram libraries gives detection numbers a boost (Searle et al 2019). Because it’s so easy to use predicted spectral libraries, I’ve been doing it for all my projects.
The tutorial above is a work in progress, so let me know if you have questions or suggestions to improve it!
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