The continued requirement to analyze larger sample cohorts to detect quantitative biologically significant differences is becoming of greater importance and placing greater demands on valuable instrument time. Traditionally, proteomic LC-MS analyses have been conducted using nanoscale chromatography in combination with data dependent analysis (DDA). However, the adoption of faster chromatography to increase sample throughput and data independent approaches (DIA) to overcome some of the shortcomings of DDA are proving increasingly popular. A number of DIA strategies with enhanced specificity exist, such as SWATH, whereby the quadrupole is stepped across a mass range of interest to increase specificity. This however can have drawbacks when utilizing faster chromatographic methods, since the duty cycle of the instrument is challenged. An alternate DIA method which also uses the quadrupole for selectivity is SONAR, whereby the quadrupole is scanned as opposed to stepping over the mass range of interest. SWATH and SONAR data were collected for a K562 cell line, demonstrating the importance of speed and specificity for accurate quantification and improved qualitative performance. When short chromatographic runs are implemented (i.e. 30 mins) a higher number of protein groups were identified for SONAR. Comparatively, 600 additional proteins were identified for SONAR when compared with SWATH. Quantitaitve gains are also observed for SONAR data showing high numbers of data points across chromatographic peak widths (typically 3 sec at half height), whilst maintaining optimum S/N. The enhanced specificity provided by the technique also demonstrates non-interfered detection of fragment ions.
Overall, results from this comparative study highlight the advantages of SONAR over SWATH based DIA methods. The implementation of a scanning quadrupole with SONAR acquisitions allow data to be acquired at a faster rate, therefore providing a higher number of protein identifications with higher quantitative precision, particularly for high throughput analyses when using microflow chromatography for example.