Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

How do we make quantitative proteomics quantitative? (#189)

Michael MacCoss 1 , Brian Searle 2 , Lindsay Pino 1 , Deanna Plubell 1 , Danielle Faivre 1 , Andy Hoofnagle 1 , Gennifer Merrihew 1 , Richard Johnson 1 , Brendan Maclean 1
  1. University of Washington, Seattle, WA, United States
  2. Institute for Systems Biology, Seattle, WA, USA

Our goal is to develop high throughput method for sampling peptides with a mass spectrometer that can be used as a quantitative measure of the phenotype.  To do this we would like a tandem mass spectrometry (MS/MS) method that can comprehensively sample all peptides in a sample continuously throughout the chromatographic elution.  MS/MS acquired using data independent acquisition (DIA) offers significant advantages in terms of selectivity, sensitivity, and dynamic range over a single stage of mass analysis.  MS/MS has significant technical advantages over MS1 analysis and we are just now in a situation where we can get high selectivity (<4 m/z), across a majority of the mass range (i.e. 400-1000 m/z), and using a rapid duty cycle (<3 sec).  That said, this does not mean that there are not substantial challenges to overcome. For example, we need methods to assess whether the peptide measurements are quantitative versus just qualitative. Additionally, global methods like proteomics struggle significantly with signal calibration -- making it difficult to compare quantitative measurements between batches, labs, and instrument platforms.  Given the prevalence of complex proteoforms we need to think carefully about what the desired outcome is of a quantitative proteomics experiment using bottom-up methodologies. Finally, while most labs feel it is important to measure as many proteins and peptides as possible, the complications associated with doing this is non-trivial -- ultimately with an increase in the number of analytes measured increases the multiple testing burden and the number of samples required to have the same statistical power.  The talk will present the current state of the art of performing quantitative proteomics using DIA. I will present use cases of what we can do, where we think the limitations are, and what work is being done to improve the methods further.