Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

Development of an optimized end to end phosphoproteomic workflow on the timsTOF PRO (#122)

Rune H Larsen 1 2 , Jarrod J Sandow 1 2 , Giuseppe Infusini 1 2 , Laura F Dagley 1 2 , Andrew I Webb 1 2
  1. Walter and Eliza Hall Institute, Parkville, VIC, Australia
  2. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia

Phosphorylation is an important post-translational modification (PTM) that occurs on proteins to modulate how intracellular signals propagate within cells in response to different stimuli or pathological conditions. Phosphoproteomics has established itself as the tool of choice to investigate the complexities of intracellular signalling cascades in an unbiased manner.

However, the utility of phosphoproteomics has been hampered by the large amount of input material normally required to enable high levels of phosphopeptide identifications and accurate quantitation (>1 mg). This limitation has restricted the majority of phosphoproteomics studies to in vitroexperiments whereas the true power of phosphoproteomics is likely to be revealed from studies of primary cells. Traditional phosphopeptide enrichment strategies have often relied on large amounts of material due to sample losses during desalting steps, lower phosphoenrichment specificity and the need to perform comprehensive fractionation which often results in reduced yields and increased technical variability. We have developed an optimised end-to-end phosphoproteomic workflow by improving cell lysis, protein digestion, phosphopeptide enrichment and identification by mass spectrometry to vastly decrease the required sample input amount. The increase in speed and sensitivity of the PASEF (Parallel Accumulation–Serial Fragmentation) acquisition strategy on the timsTOF Pro (Bruker) has greatly aided the identification of phosphorylated peptides without the need for extensive fractionation.

We have optimised collision energies, gradient times and trapped ion mobility spectrometry (TIMS) settings in order to refine the PASEF strategy for ultrasensitive phosphoproteomics. Using our end-to-end phosphopeptide enrichment workflow, we demonstrate the capabilities of the timsTOF by enabling robust phosphopeptide quantitation with minimal starting material.