Offset Mass Carrier Proteome Improves Quantification of Multiplexed Single Cell Proteomics

Multiplexed single cell proteomics by mass spectrometry (scpMS) approaches currently offer the highest throughput as measured by cells analyzed per day. These methods employ isobaric labels and typically a carrier proteome, a sample added at 20 to 500× the single cell level that improves peptide sampling and identification. Peptides from the carrier and single cell proteomes exist within the same precursor isotopic cluster and are co-isolated for identification and quantification. This represents a challenge as high levels of carrier proteome limit the sampling of peptide ions from single cell samples and can potentially lead to decreased accuracy of quantitative measurements. Here, we address this limitation by introducing a triggered by offset mass acquisition method for scpMS (toma-scpMS) that utilizes a carrier proteome labeled with nonisobaric tags that have the same chemical composition but different mass as the labels used for quantitative multiplexing. Within toma-scpMS, the carrier proteome and single cell proteome are separated at the precursor level, enabling separate isolation, fragmentation, and quantitation of the single cell samples. To enable this workflow, we implemented a custom data acquisition scheme within inSeqAPI, an instrument application programming interface program that performed real-time identification of carrier proteome peptides and subsequent triggering of offset single cell quantification scans. We demonstrate that toma-scpMS is more robust to high levels of carrier proteome and offers superior quantitative accuracy as compared to traditional multiplexed scpMS approaches when similar carrier proteome levels are employed.