Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics.

A recent study demonstrated a substantial increase in the peptide signal and corresponding proteome coverage when employing 0.5% acetic acid (AA) as the ion pairing modifier in place of the 0.1% formic acid traditionally used in shotgun proteomics. Given the strictly limited material and counterintuitive observations by others in the emerging field of single-cell proteomics, we chose to investigate this alternative modifier in the analysis of subnanogram proteome dilutions. When digest standards as low as 20 pg total load on the column were evaluated, AA led to increased proteome coverage at every peptide load assessed. Relative improvements were more apparent at lower concentrations, with a 20 pg peptide digest demonstrating a striking 1.8-fold increase to over 2000 protein groups identified in a 30 min analysis. Furthermore, we find that this increase in signal can be leveraged to reduce ramp times, leading to 1.7× more scans across each peak and improvements in quantification, as measured by %CVs. These results can be reproduced on multiple trapped ion mobility instruments. When evaluating single cancer cells, approximately 13% more peptide groups were identified on average when employing AA in the place of FA. These results suggest that ion pairing modifiers and other additives warrant re-evaluation in the context of low-input and single-cell proteomics. All vendor raw and processed data are available through ProteomeXchange as PXD046002 and PXD051590.