Introduction:
In the realm of proteomics, accurate and precise relative protein quantitation is the key to unravel the complex secrets of biological processes. Dia-PASEF is an advanced variant of DIA, capitalizing on the additional dimension of separation unlocked on the timsTOF platform by trapped ion mobility separation (TIMS). Demonstrating these capabilities resulted in optimizing a dia-PASEF acquisition method to analyze both single proteome samples and complex hybrid proteome mixtures for benchmarking.
Methods:
Tryptic digests of a HeLa, yeast and E. coli were either loaded directly on column or combined in defined ratios.
Samples were loaded on a 15cm C18 column (75μm, 1.6μm, Aurora, IonOpticks) using a nanoElute 2 nano HPLC (Bruker) coupled to a timsTOF HT (Bruker) via a CaptiveSpray 2 ionization source (Bruker) using a 15-min ACN gradient. For the dia-PASEF acquisition, a window placement scheme optimized via the py-diAID tool was used.
Data were processed in Spectronaut (v18, Biognosys) using directDIA+™.
Results:
In our study we were able to cover nearly the complete yeast proteome by identifying on average 4408 protein groups using a 15 min gradient. 95% of the protein groups were identified and quantified with CV values below 20% from triplicate injections.
When analyzing a human protein digest sample nearly 105,000 stripped peptide sequences from close to 8000 protein groups have been identified with excellent reproducibility.
For evaluation of the presented setup for quantitative proteomics we used samples mixed in defined ratios (HeLa, yeast, E.coli). Within a 15 min gradient we could identify and quantify on average 12,893 protein groups from 173,851 peptide precursors. The measured ratios obtained for human, yeast and E.coli were close to the expected ratios.
Conclusions:
dia-PASEF on the timsTOF HT enables high proteome coverage and accurate quantitation in short gradients of 15 minutes.