Bottom-up protein identification has always been one of the most demanding tasks for biological MS experiments, requiring a combination of peak capacity, speed and sensitivity to sequence as many peptides as possible from a highly complex mixture of digested proteins. The unique Trapped Ion Mobility Spectrometry (TIMS) capability of the timsTOF Pro adds an extra dimension of separation, resulting in unprecedented peak capacity and sensitivity and enabling the PASEF (Parallel Accumulation Serial Fragmentation) workflow for > 100Hz MS/MS with no sensitivity or resolution loss, all while operating at 100% duty cycle.

In a typical shotgun proteomics experiment, peptides from more than a million proteoforms, covering up to 10 orders of magnitude in concentration are separated by nano-LC typically using a 2 hour gradient. This represents a significant challenge for the mass spectrometer which must detect, isolate and fragment as many of the millions of resulting peptides as possible. To generate extensive protein identification information, adding ion mobility as an extra dimension of separation, has the potential to dramatically increase peak capacity. However, this must not be at the detriment of speed or sensitivity.

The timsTOF Pro with PASEF leads to exceptional protein identification performance and depth of analysis. When coupled with the nanoElute nano-flow UHPLC and CaptiveSpray ion source the timsTOF Pro can routinely identify more than 5000 protein groups from 200 ng of an HeLa cell digest separated with a 90 min gradient.

The phenomenal speed can be used to reduce the gradient time and increase throughput while maintaining unprecedented depth of analysis. Close to 4000 protein groups can be identified from 100 ng of a Hela cell digest separated with a 30 min gradient.

PASEF finally allows clinical proteomics to become a reality. The combination of the system’s speed and sensitivity reproducibly identifies over 1400 Hela cell protein groups from a 50 ng injection separated with a 5 min gradient.