Background: With the previously introduced “Parallel Accumulation Serial Fragmentation” method (PASEF, Meier et al., JPR 2015, PMID: 26538118) for ion mobility (IMS) quadrupole time of flight (QTOF) instruments, five to ten times faster data dependent acquisition of fragment ion spectra became possible. This approach requires a fast, two-dimensional precursor selection algorithm, using mass as well as ion mobility information.
Methods: In PASEF mode, peptide ions elute from the IMS device as a condensed package. For most efficient MSMS acquisition, the quadrupole isolation window needs to switch its isolation position exactly synchronized to these elution times in the fastest possible order. The corresponding algorithm was developed and evaluated using tryptic digests of human HeLa cell lysates, separated by 90min nanoLC gradients. Data were analyzed using DataAnalysis(Bruker), Mascot(www.matrixscience.com), and MaxQuant (Cox group, MPI of Biochemistry).
Results: The precursor selection algorithm detects m/z and mobility positions of all precursors in the MS1-IMS scan. Then they are scheduled for measurement across multiple (ten to twenty) consecutive IMS experiments aiming for the most efficient utilization of measurement time. Low intensity precursors are measured multiple times to achieve sufficient spectra quality. Elution length of an individual precursor ion is dependent on IMS resolution, a function of mobility scan time. For tryptic peptides, mobility scan times of 25, 50, 100 and 200 ms resulted in average mobility resolutions between 20 and up to 80 and elution lengths between 1.8 and 10.6 ms. With a 100ms IMS separation up to 900 000 individual PASEF MSMS spectra can be acquired during a 90 min HeLa nanoLC-run, resulting in about 200 000 unique MSMS spectra after combining repetitive measurements.
Conclusion: We developed a 2D-precursor selection algorithm for PASEF that determines and schedules precursors in complex samples within 100-200 ms, well suited for an LC timescale.