Improved ETD Performance for Top-Down Applications

Electron transfer dissociation (ETD) is an effective strategy for analyzing peptides and proteins, particularly those with post-translational modifications or multiple precursor charge states. In order to get the best results, researchers adopting this strategy for top-down proteomics must first find ways to overcome a low spectral signal to noise (S/N) ratio. One group of scientists, Mullen et al., recently investigated ways to overcome this issue by changing the pre-reaction (starting) locations of the precursor and reagent and the order in which they are injected into the high pressure cell (HPC) of the dual cell linear ion trap (LIT).¹

The team reports that previous attempts to increase the S/N ratio relied on a multiple-fill approach by accumulating products of multiple ETD reactions and analyzing them collectively in a single m/z analysis. They concluded that this type of strategy requires a longer scan time, and they decided to instead extend the per-fill precursor capacity of the linear ion trap and include new ETD scan functions and a LIT with an extended front section.

For their experiments, the scientists used a Thermo Scientific Orbitrap mass analyzer from a hybrid quadrupole LIT instrument. They evaluated the maximum number of precursor charges retained after sequestration and the maximum achievable reaction rate for five different scan functions for both HPC configurations:

• Reagent Center – Precursor Front (RC, PF)

• Precursor Back – Reagent Center (PB, RC) [“Standard”]

• Precursor Center – Reagent Front (PC, RF) [“Enhanced”] 

• Precursor Back – Reagent Front (PB, RF) 

• Reagent Back – Precursor Center (RB, PC)

Mullen et al. were able to extend dynamic range in the ETD spectra of large protein molecules with a three-fold or greater improvement in precursor capacity. They saw an improved S/N ratio with both the extended front section LIT and the standard LIT. They also found the extended LIT was able to maintain pseudo first order kinetics longer than the standard counterpart due to the increased reagent capacity of the front section.

Since ETD is widely used for characterizing post-translational modifications, the benefits of an improved performance will be a great asset to researchers looking for greater proteomic coverage. 

 

Reference

1. Mullen, C., et al. (2015) “Considerations for attaining improved ETD performance for top down applications,” Thermo Scientific.