Shotgun Protein Identification

Depth and speed: a breakthrough for shotgun proteomics

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.

The TIMS-PASEF^® solution

A complexity issue
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.

TIMS-PASEF^®
The unique design of TIMS enables the PASEF^® method where concentrated packets of ions elute from the ion mobility cell with widths of 2-5 ms enabling MS/MS acquisition speed >100 Hz and a duty cycle that approaches 100%. The extra separation is thus obtained on top of an increase in speed and sensitivity, while the TOF analyzer allows to maintain an ultra-high MS/MS resolution at all MS and MS/MS speeds.

Parallel accumulation serial fragmentation (PASEF®). PASEF® strategy in a timsTOF Pro instrument where ions are accumulated while the second TIMS region is serially eluting peptide species.

PASEF^® for deep proteomics studies

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.

PASEF^® for increased throughput in shotgun proteomics
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.

Standard PASEF® method: a 1.1 second cycle time containing 10 TIMS separation event. On average, 12 MS/MS are performed in each TIMS separation event.

PASEF^® for increased protein identification sensitivity

The remarkable sensitivity increase that derives from the TIMS separation enables new possibilities for low concentration samples. Using the standard PASEF^® method, with additional tuning, 12 ng of a Hela cell digest separated with a 60 min gradient identified over 2900 protein groups.

The ideal clinical proteomics companion
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.

Label-free quantification outcome of a three-proteome mixture. The total protein load is 150ng, separated with a 90min gradient. MaxQuant was used for processing.