A deeper look at carrier proteome effects for single-cell proteomics

Multiplexing approaches using tandem mass tags with a carrier proteome to boost sensitivity have advanced single cell proteomics by mass spectrometry (SCoPE-MS). Here, we probe the carrier proteome effects in single cell proteomics with mixed species TMTpro-labeled samples. We demonstrate that carrier proteomes, while increasing overall identifications, dictate which proteins are identified. We show that quantitative precision and signal intensity are limited at high carrier levels, hindering the recognition of regulated proteins. Guidelines for optimized mass spectrometry acquisition parameters and best practices for fold-change or protein copy number-based comparisons are provided. Ye et al. address a highly debated aspect of many recent single cell proteomics studies, the extent and impact of the carrier proteome. Through deployment of a mixed-species sample at various spike-in levels, they set up an experimental system for further exploration of the carrier effects on TMT-based quantification of ultra-low input samples.