Targeted proteomics approaches are now commonly used, either to perform targeted biomarker candidate discovery or to validate candidate biomarkers that had been pointed out by untargeted discovery proteomics approaches. In this study, we are performing a first evaluation of the potential of the recently introduced nano-ESI tims Q-TOF architecture for targeted proteomics approaches. More specifically, we want to check if the time and space concentration of ions that results of the Trapped Ion MobilitySpectrometry (tims) separation process can translate in to increased sensitivity and specificity performances for targeted proteomics approaches.
An equimolar mixture of 259 quantified synthetic peptides labelled with stable isotopes (AQUA) was diluted in a 100ng/μl human cell line digest. The dilution series covered 6 concentration levels ranging from 31.25 amol/μl to 25 fmol/μl. All samples were separated by nano-HPLC with a 60 min gradient and analysed on a high-resolution timsTOF Pro instrument (Bruker Daltonics) operated in data dependent PASEF mode or an exploratory targeted TIMS-PRM acquisition modes. The global sensitivity, selectivity and detectability of the different acquisition modes was evaluated with the latest version of the Skyline Ô software.
The PASEF acquisition of cell lysate digest spiked with 25fmol, 6,25fmol, 1562.5amol, 500amol, 125amol and 31.25 amol of the AQUA mixture allowed to identify 253/235/205/104/48 and 4 of the original 259 AQUA peptides, respectively. Using an exploratory tims-PRM approach with a 100 ms tims trapping time, and prior to any collision energy optimization, 110/168/205/213 and all of the AQUA peptides could be quantified at a the 31/125/500/1562/6250 amol level, respectively. Increasing the tims trapping time allowed to increase the detected S/N ratio. The results obtained after a more complete optimization will also be presented.
We have demonstrated a real (yet) unexploited potential of the tims-Q-TOF architecture for targeted proteomics approaches.