Quadrupole time-of-flight (Q-ToF) MS is a well established tool for both discovery and quantitative applications. A new mode of DIA operation, SONAR, was recently introduced whereby a low-resolution quadrupole is scanned repetitively over alternating low (MS1) and elevated energy (MS2) scans, producing data of a similar format as Ion mobility enabled modes by utilising the same multi-dimensional acquisition system. The elevated collision energy profile is applied to the gas cell in a linear fashion and corresponding to the full mass scale and mass position of the quadrupole. As such, optimisation of the profile is an important consideration in order to produce data of sufficient quality for qualitative identification tools.
Samples were introduced to the Xevo G2XS Q-ToF Mass Spectrometer by either direct infusion, Acquity LC, micro or nanoscale LC. The quadrupole mass filter of the MS was characterised to allow various transmission windows to be employed for different molecule classes; peptides, small molecules (metabolites and lipids) and intact proteins. Initially, the full m/z range of the quadrupole was continuously and repetitively scanned and the MS2 CID energy applied in a linear fashion to correspond with the quadrupole mass position. Further optimisation of the collision energy can be achieved by segmenting the quadrupole m/z scale into multiple steps. The 2D data format was processed using both commercial (e.g. PLGS, UNIFI, Progenesis QI(p), Mascot, SimLipid, Spectranout, etc.) and open source (Skyline, OpenMS, OpenECHO) software and Driftscope for viewing multidimensional data.
Initial experiments were carried out to determine optimum collision energy ramps using one quadrupole mass range, for example m/z 400 – 900 for analysis of complex peptide mixtures and m/z 500 – 1200 for small molecule applications. These were carried out in a systematic manner, i.e. by altering the starting and ending collision energy values in 5 V steps. Optimum ramps, based upon peptide identification rates and feature identifications, showed that ramps of 14 to 40V for proteomic and 20 to 50 V (+ve) and 25 to 55 V (-ve) for small molecules were appropriate values. However, the potential to further optimize collision energy ramps, with the aim of maximizing coverage, based upon precursor m/z and retention time is apparent when analyzing the identified precursors from the injection of 6 µg K562 cell line onto a 300 micron ID column. It is apparent there are distinct retention time regions where SONAR experiments would potentially benefit from using multiple quadrupole m/z ranges and collision energies, i.e. retention order, dependent. The analysis of complex proteomics samples show that the multi-step method has the potential to increase coverage and subsequent quantitation by > 20%. We also applied this same methodology to small molecule (lipidomics) experiments, increasing the feature identification rate significantly and improving the qualitative information by extraction of analyte class information based upon neutral loss or product ion extraction.