Mass spectrometry approaches to single cell proteomics (SCP) are gaining momentum as sample preparation methods and mass spectrometry analysis become more sensitive. One approach to SCP labels single cells with isobaric tags and includes a “carrier proteome” at relatively high excess to provide enough ions for peptide identification. Single cells are then quantified utilizing reporter ions 100-200x lower in intensity than the highest multiplexed channel. The carrier proteome channel helps in identification, but makes quantification of the experimental channels more challenging due to the limited dynamic range of the Orbitrap mass analyzer.
Here we introduce an offset mass triggered data acquisition approach that allows a carrier proteome channel to be used without impacting the intra-scan dynamic range of the Orbitrap mass analyzer. This inter-cluster scheme moves the “carrier proteome” peptides into a separate precursor isotopic cluster by utilizing super-heavy TMT (SH-TMT). The inter-cluster approach enables isolation of the experimental peptides without the isolation of the “carrier proteome” and ensures that all resulting reporter ions will be used for quantitation of experimental ratios. Using a recently described multiplexed targeted approach (TOMAHAQ) we explore the impact of the carrier proteome in the context of this inter-cluster approach and typical SCP experimental designs. We then demonstrate how a triggered offset mass approach could be implemented in data dependent acquisition, enabling it to be easily used in SCP experiments.
From these data we make recommendations for the utilization of carrier proteomes and experimental design of SCP experiments, as well as highlight key quantitative metrics (e.g., reporter ion signal-to-noise, injection time, etc.) to ensure meaningful biological results.