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.