Most Data Independent Acquisition strategies utilize a stepped modes of acquisition, where the first mass filter is set to pass a fixed m/z range, with windows typically 5-20 m/z unite. The need to use a number of different steps to cover the m/z range of interest leads to a relatively long duty cycle of acquisition, which slows the analysis time and can lead to relatively poor quantitative reproducibility. In SONAR, a resolving quadrupole is scanned repetitively over alternating low and elevated energy scans. This produces data in a similar format to Ion Mobility enabled acquisitions. The relatively high speed duty cycle can be exploited to reduce analysis time and thus sample throughput. Notably, SONAR can be used with very high throughput separation systems. An additional feature of SONAR it that both conventional database searching and spectral library matching can be used to make qualitative identifications. We have applied SONAR to address several biomedical projects – changes in the synaptosome as a function of gene knock-down, and the effects of drug treatments on protein:protein complexes.
The fast duty cycle of SONAR has been shown to provide very high quantitative reproducibility, via the analysis of project-specific Study Pool QC samples. For example, in the synaptosome study, 1,712 proteins gave an overall 5.8% CV, whereas the protein:protein interaction QC pools gave 7.49% CV. In addition to the high quantitative reproducibility, these experiments show SONAR provides information-rich datasets, with a qualitative and quantitative dataset acquired for each sample. Since SONAR does not require a DDA spectral library, the datasets can be directly searched against a FASTA database.