Protein identification header

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. Trapped Ion Mobility Spectrometry (TIMS) 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.

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 peptides as possible. 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 TIMS-PASEF solution

The unique design of TIMS enables the PASEF method where concentrated packets of ions elute from the ion mobility cell with widths of 2-5ms enabling MSMS acquisition speed >100Hz and a duty cycle that approaches 100%.

TIMS analyzer
ions are trapped in TIMS analyzer 1 while they are eluted from TIMS analyzer 2,resulting in a duty cycle close to 100%. Accumulation in analyzer 1 lasts 100 ms, but each ion species is eluted from analyzer 2 in a 5ms width ion packet. This space and time concentration is key to enable high-speed and high-sensitivity analysis.
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 deep proteomics studies

The timsTOF Pro with PASEF leads to exceptional proteomics performance and depth of analysis. When coupled with the nanoElute nano-UPLC and CaptiveSpray ion source the timsTOF Pro can routinely identify more than 5,000 protein groups from 200 ng of an HeLa cell digest separated with a 90 min gradient.

PASEF for increased throughput

The phenomenal speed "seen on the timsTOF Pro" 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 100ng of a Hela cell digest separated with a 30 min gradient.

PASEF for increased protein sensitivity

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

PEAKS database search results
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.

The ideal clinical proteomics companion

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

For Research Use Only. Not for Use in Clinical Diagnostic Procedures.