Enhancing Tandem MS Sensitivity and Peptide Identification via Ion Preaccumulation in an Orbitrap Mass Spectrometer.

High-throughput mass spectrometry-based proteomics has gained increasing interest for both academic and industrial applications. As implementation of faster gradients has facilitated higher sample throughput, mass spectrometers must adapt to shorter analysis times by enhancing the scanning speed and sensitivity. For Orbitrap mass spectrometers, faster scan rates are constrained by the need for sufficient ion accumulation time, particularly given the limitations on the duty cycle at high repetition rates, and transient length, which determines analyzer sensitivity and resolving power. In this context, implementing alternative ion scheduling and better ion signal-processing strategies is needed to unleash the speed of these instruments. Here, we introduce a new scanning strategy termed preaccumulation, which enables the storage of ions in the bent flatapole in parallel to the operation of the C-trap/IRM, leading to a significant improvement in ion beam utilization and enabling for the first time scanning speeds of ∼70 Hz on hybrid Orbitrap instruments. The combination of preaccumulation and increased scan speeds notably enhances peptide and protein group identifications for short LC gradients and improves sensitivity for high-throughput applications. These benefits were further amplified when coupled with the full mass range phase-constrained spectrum deconvolution method (ΦSDM), especially for fast, lower-resolution Orbitrap measurements with short LC gradients. Overall, we demonstrate that preaccumulation of ions in the bent flatapole offers distinct advantages, particularly for conditions with reduced signal input. Since no hardware changes are required, this approach is highly attractive for Orbitrap mass spectrometers operated with fast MS/MS acquisition methods.