Basics of targeted mass spectrometry with Skyline

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:

  1. Assess the experimental pros and cons of targeted proteomics, and compare to discovery proteomics.
  2. Explain the fundamentals of mass spectrometry proteomics, including peptide fragmentation and basic components of a mass spectrometer
  3. Describe the steps of a targeted proteomics workflow and the information required to build an assay
  4. Apply the Skyline software ecosystem to their own targeted proteomics experiments.

Throughout the lecture-based workshop, I mixed participant question-and-answer and examples of the concepts discussed in Skyline. Finally, I closed with three hands-on examples of using Skyline to build a Parallel Reaction Monitoring (PRM) mass spectrometry experiment.

You can check out the recording below:

Using Prosit predicted spectral libraries to build GPF chromatogram libraries

UPDATE: I learned that Searle et al 2020 includes a tutorial in the Supplementary Note 1!

Click here to go to my version of the tutorial

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 2020). 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!

Resources for beginner bioinformatics


Learning to code, coming from an experimental background, can be a frustrating and intimidating experience. Although there are many free courses online that will teach you the basics of Python and programming, the best way to improve is to simply practice. The best practice is a project you are personally motivated and passionate about. For graduate students, this might be a component of your thesis or a side project that complements your research interests. Coming up with an interesting project might be daunting at first, however, so here are some resources for quick, achievable practice problems to help keep you coding.

Practice mathematical/computer programming problems
Python basics with
Beginner bioinformatics with

Additional resources:
Check your code style online