Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Optimizing the isolation width in Orbitrap instruments to maximize the number of label-free quantified peptides and protein. (#492)

Carmen Paschke 1 , Tabiwang N. N. Arrey 1 , Rosa Rakownikow Jersie-Christensen 1 , David Horn 2 , Martin Zeller 1 , Romain Huguet 2 , Bernard Delanghe 1
  1. Thermo Fisher Scientific (Bremen) GmbH, Bremen, BREMEN, Germany
  2. Thermo Fisher Scientitic , San Jose, CA, USA

In recent years, the performance of the filtering quadrupole in Orbitrap instruments has been improved. Isolation widths down to 0.4 Th are available. For reporter based quantification, smaller widths should be used to avoid interference from the co-isolated peptides. For Label Free Quantification (LFQ) that might not be the case. Here we will investigate the optimal isolation width for maximizing the number of identified and quantified proteins and peptides, together with the best possible accuracy and precision in LFQ experiments on Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ and Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometers.

To optimize identification and quantification parameters for LFQ, HeLa digests have been analyzed in different concentrations using different isolation widths on a modified orbitrap tribid instrument and a modified quadrupole orbitrap.

For precision and accuracy experiments yeast digest was used in different amounts in a constant background of 200 ng HeLa digest.

Identification strategies using SequestHT with and without second peptides identification and spectral libraries have been used.

Data analysis was performed with a beta version of Thermo Scientific Proteome Discoverer™ 2.4 software.