Session: Session 3

Improved proteome coverage combined with reproducible quantitation on the timsTOF platform

Introduction  

In the realm of proteomics, accurate and precise relative protein quantitation is the key to unravel the complex secrets of biological processes. In recent years there has been a shift towards analyzing complex proteomics samples in shorter time to keep up with the increasing demand of sample throughput. Nowadays Data-Independent Acquisition (DIA) is widely used. Dia-PASEF is an advanced variant of DIA, capitalizing on the additional dimension of separation by trapped ion mobility separation (TIMS). We applied more advanced (py_diAID, vistaScan) window schemes to evaluate their performance on complex proteomics samples analyzed from different amounts in short gradient times.  



Methods

Tryptic digests of a human cell lysate, Saccharomyces cerevisiae and E. coli  were loaded on a 15cm C18 column and separated using a 15 min gradient  using a nanoElute 2 nano HPLC (Bruker) coupled to a timsTOF HT (Bruker) operated in dia-PASEF mode  (Bruker).  

Data were processed in Spectronaut (v19, Biognosys) using library-free mode with human, E. coli, and yeast Uniprot fasta files. False discovery rate (FDR) was controlled at 1% for peptide and protein group level. 



Results

Samples mixed in defined ratios (HeLa, yeast, E. coli) were used to evaluate the setup for quantitative proteomics. Within a 15 min gradient we could identify and quantify 12,893 protein groups from 173,851 peptide precursors from 400ng sample.Investigation of the quantitative performance showed low median coefficient of variation for the replicate runs at around 5% on protein group level. The chosen experimental design also enabled the evaluation of the quantitative accuracy with pre-determined theoretical ratios. The measured ratios obtained for human, yeast and E. coli were close to the expected ratios with low levels of standard deviation.



Conclusion

Sophisticated dia-PASEF methods enable high proteome coverage and accurate quantitation in short gradients of 15 minutes on the timsTOF platform.