Spectronaut 14 fully supports Bruker’s dia-PASEF as well as Thermo FAIMS technologies, offering a convenient way to use these new acquisition modes and make the best use of them to get better proteome coverage than ever before.
Spectronaut 14 adds full support for dia-PASEF analysis. You can analyze dia-PASEF runs either using a DDA (PASEF) based spectral library or directDIA (Figure). Pulsar, our database search engine, can generate an ion-mobility-enhanced library from PASEF or dia-PASEF runs. For the targeted analysis of dia-PASEF data, Spectronaut introduces a novel high-precision ion mobility workflow [ASMS 2020, MP-115] which is similar to our workflow for retention time analysis [Bruderer, 2016]. Additionally, the time it takes to create PASEF based spectral libraries has been almost cut in half.
Three species were mixed in different ratios, in two samples using human as the background [Kaspar-Schoenefeld, 2019]. Samples were measured using timsTOF Pro in dia-PASEF mode on a 100 min gradient. The measurements were analyzed using directDIA (i.e. without using any library-specific runs). In total, 7’434 protein groups were identified on average. Label-free quantification reflects the expected ratios of the species.
We significantly improved our support for FAIMS Pro™ devices with ion mobility filtering [ASMS 2020, MP131]. DIA methods with FAIMS usually come in two main flavors: Single compensation voltage (CV) methods and multi CV methods. Both of these methods are now supported. Pulsar, our database search engine, can create a compatible library both from FAIMS DIA and DDA runs.
DIA data for triplicate runs of HeLa cell samples were recorded on a Thermo Orbitrap Exploris 480 using a 120-minute gradient, with and without FAIMS Pro™ (three CVs). Data analysis was done using project-specific hybrid libraries [Muntel, 2019]. On average, we saw a 14% increase in modified peptide identifications and a 34% increase in protein group identifications per file when using FAIMS Pro™.