Background
The key challenges often faced by mass spectrometry users include insufficient sensitivity and the inability to maintain uninterrupted and optimal operation of the instrument for extended periods. A new Q-TOF platform has been developed to address the above needs and accelerate the path to results.
Methods
A commercial human K562 tryptic digest was diluted and analyzed using microflow and nanoflow reverse-phase chromatography on a Waters M-Class HPLC and EvoSep One system with varying gradient lengths. Analysis was performed using a novel quadrupole time-of-flight (Q-TOF) mass spectrometer with Zeno SWATH DIA and ZT Scan 2.0 DIA. Data were processed using PEAKS software or DIA-NN.
Results
The K562 tryptic digest shows that the signal intensity of the novel QTOF platform was approximately 3-5 times higher than that of the predecessor system for an equivalent on-column loading. Higher sensitivity resulted in significant increases (>200%) in the number of quantifiable peptides and proteins, mostly at low sample loadings and with high-throughput methods (200, 300 and 500 SPD). For a 50 ng K562 sample analyzed with 200-500 SPD methods on an EvoSep One system and ZT Scan 2.0 DIA, 4800-6400 protein groups (4200-5900 <20% CV) and 32,000-51,000 precursors were identified with DIA-NN or 40,000-64,000 precursors with PEAKS. For a 5 ng load at 200-500 SPD, 3900-5000 protein groups were identified (3000-4100 <20% CV) corresponding to 26,000-36,000 precursors or 29,00-38,000 with PEAKS. For a 250 pg sample loaded with a 30 min nanoflow gradient, over 4900 protein groups and 35,000 precursors were detected. Software enhancements will enable users to have higher confidence in optimal MS operation, with optimized and guided tuning capabilities, as well as track system performance by monitoring calibrations longitudinally via a graphical plotting interface and statistical analysis that triggers notifications.
Conclusions
The next-generation Q-TOF platform offers improved sensitivity, performance, and software enhancements.