Introduction
prm-PASEF is a targeted acquisition method that fully exploits the multiplexing capability and the high resolution of TIMS-Q-TOF mass spectrometers. Multiple peptides can be sequentially measured from a single ion mobility scan without compromising the sensitivity. We evaluated the reproducibility, sensitivity, accuracy and dynamic range of the method by using aqua peptides spiked in a Hela cell line digest and a depleted serum samples digest. Finally, we applied the method to quantify the mutations and isoforms of the Ras oncoproteins family in cancer cell lines.
Methods
The quantitative performance of prm-PASEF was evaluated with a tryptic digest of HeLa cells and depleted human serum samples spiked with 201 AQUA peptides and 15 light peptides. Serial dilutions ranging from 5.5 to 50,000 amole/µl were generated with 15 heavy/light peptides pairs. The other 186 AQUA peptides were spiked at the constant concentration of 2 fmole/µl. Ten cancer cell lines were analyzed for screening the Ras mutations. All acquisition were performed on a timsTOF Pro instrument.
Results :
The Aqua peptides calibration curves showed a signal response fitted by a linear regression over a concentration factor of 2900 (from 17.2 to 50,000 amole injected column), and an averaged RSD of 3% for the heavy/light peptide signal ratios. For label free quantification, the 2 fmole 186 AQUA peptides measured over 30 LC-MS runs showed a median RSD of 10.5% and a median of 25 data points across the chromatographic peaks. Finally, the G12 mutations of Ras protein family and the three isoforms (NRas, KRas and HRas) were identified and quantified in 10 colon and lung cancer cells lines using a 10 min chromatography separation.
Conclusions
We developed a new targeted acquisition method that takes advantages of trapped ion mobility and fast high-resolution Q-TOF. The method is well suited for clinical applications that require to measure high density of targets with fast chromatography separations.