Parallel Accumulation-Serial Fragmentation (PASEF): Multiplying Sequencing Speed and Sensitivity by Synchronized...

Area of Science:

• Proteomics
• Analytical Chemistry
• Mass Spectrometry

Background:

• Liquid chromatography-mass spectrometry (LC-MS) often faces limitations with simultaneous precursor elution.
• Data-dependent acquisition discards many potential precursors during analysis.
• Existing methods struggle to efficiently fragment co-eluting peptides.

Purpose of the Study:

• To overcome the limitations of discarding co-eluting precursors in LC-MS proteomics.
• To develop a method for serial selection and fragmentation of multiple precursors in a single scan.
• To increase the speed and efficiency of shotgun proteomics analyses.

Main Methods:

• Implementation of trapped ion mobility spectrometry (TIMS) on an orthogonal quadrupole time-of-flight (QTOF) mass spectrometer.
• Development of synchronized quadrupole scans with sub-millisecond switching times.
• Introduction of parallel accumulation-serial fragmentation (PASEF) for precursor selection and fragmentation.

Main Results:

• Demonstration of serial selection and fragmentation of multiple precursors within single 50 ms TIMS scans.
• Achieved hundreds of MS/MS events per second with full sensitivity using PASEF.
• Estimated a 10-fold gain in sequencing speed for shotgun proteomics without sensitivity loss.

Conclusions:

• PASEF overcomes the precursor limitation in LC-MS-based proteomics.
• The method enables high-throughput, sensitive analysis of complex proteomes.
• PASEF represents a significant advancement in mass spectrometry-based proteomics speed and efficiency.