Introduction

Single-cell and low-input proteomics leverage powerful new tandem mass spectrometry (MS/MS) tools for exploring cell biology at a granular level. While improvements in instrumentation and sample preparation methodologies have made single-cell proteomics attainable by many labs, consistently detecting peptides in single-cell experiments remains a challenging endeavor. One way to improve our ability to detect peptides in low-input experiments is to use reference spectra acquired from bulk samples with more input material. Recent reports have suggested that the quality of MS/MS spectra may change as input material decreases, and in this work, we sought to determine how many ions are required for an Orbitrap^TM (OT) and Astral^TM (AST) mass analyzer to produce internally consistent spectra using the same instrument.

Preliminary Data

Comparisons of low-input and bulk proteomics spectra rely on using different acquisitions for each sample type. In some cases, these measurements have been performed in different labs on instruments with different configurations and tunings. Even when performed on the same instrument, changes in sample complexity and composition affect what background ions can interact with peptides, potentially changing ion interference. For this work, we designed a DIA method to assess the influence of the number of ions on the quality of MS/MS spectra in an OT within a single injection. Rather than modulate sample concentrations to change the number of ions in the IRM, we adjusted AGC to mimic low-input level measurements by limiting the number of ions. This gave us a unique platform to test the effects of limited ions on the quality of MS/MS, where higher AGC spectra act as controls. Integrating the precursor isolation window from preceding precursor spectra, we calculated the number of ions within each MS/MS measurement, given the ion injection time. Within each packet, we calculated the spectral contrast angle between the optimal MS/MS (1000% AGC) and MS/MS with fewer ions limited by lower AGC targets. We observed that the quality of MS/MS spectra measured in the OT was tied to the ion count of the highest fragment, suggesting that the relationship is governed by ion statistics, rather than total ion count. We found the OT required approximately 150 ions in the OT as the predominant fragment to produce >80% spectral angle. This corresponded to approximately 1400 total precursor ions, equivalent to only 1.4% of the normalized AGC target in MS/MS analysis in most low-input DIA experiments. Consequently, we suggest that optimal acquisition parameters e.g. longer IIT and higher OT resolving power can improve MS/MS spectra quality in single-cell measurements and eliminate differences, observed from the bulk analysis.