Parallel Accumulation–Serial Fragmentation (PASEF): Multiplying Sequencing Speed and Sensitivity by Synchronized Scans in a Trapped Ion Mobility Device

TLDR

LC‑MS‑based proteomics often elutes many precursors simultaneously, yet data‑dependent acquisition fragments only one at a time, discarding the rest. The authors aim to eliminate this limitation by integrating trapped ion mobility spectrometry (TIMS) with a quadrupole time‑of‑flight mass spectrometer. TIMS accumulates all precursor ions in parallel and releases them sequentially according to ion mobility, while synchronized sub‑millisecond quadrupole scans target precursors from a top‑N list for serial fragmentation. The resulting PASEF method serially fragments multiple precursors within a single 50‑ms TIMS scan, achieving hundreds of MS/MS events per second at full sensitivity and predicting a ten‑fold increase in sequencing speed without loss of sensitivity.

Abstract

In liquid chromatography-mass spectrometry (LC-MS)-based proteomics, many precursors elute from the column simultaneously. In data-dependent analyses, these precursors are fragmented one at a time, whereas the others are discarded entirely. Here we employ trapped ion mobility spectrometry (TIMS) on an orthogonal quadrupole time-of-flight (QTOF) mass spectrometer to remove this limitation. In TIMS, all precursor ions are accumulated in parallel and released sequentially as a function of their ion mobility. Instead of selecting a single precursor mass with the quadrupole mass filter, we here implement synchronized scans in which the quadrupole is mass positioned with sub-millisecond switching times at the m/z values of appropriate precursors, such as those derived from a topN precursor list. We demonstrate serial selection and fragmentation of multiple precursors in single 50 ms TIMS scans. Parallel accumulation–serial fragmentation (PASEF) enables hundreds of MS/MS events per second at full sensitivity. Modeling the effect of such synchronized scans for shotgun proteomics, we estimate that about a 10-fold gain in sequencing speed should be achievable by PASEF without a decrease in sensitivity.