Advanced Data Acquisition Approaches to Multiplexed Single Cell Analysis

Single cell proteomics was revolutionized by the introduction of SCoPE-MS, a method that utilizes a carrier proteome added at 20x to 500x single cell channels to enable peptide detection, selection, and quantification. Recently, it has been demonstrated that adding excess carrier proteome may increase measurement variability as well as decrease measurement accuracy and dynamic range. This can be overcome by decreasing the amount of carrier proteome or increasing the number of ions sampled - but quantitative analysis is impacted at some level by the presence of carrier proteome reporter ions. Here, we introduce two advanced data acquisition approaches that utilize a carrier proteome labeled with an offset mass chemical tag (i.e., TMT-SH) that separates the carrier proteome peptide cluster from the multiplexed single cell proteomes and enables quantification of single cells in the absence of carrier proteome reporter ions. The first approach SureQuant-TMT enables the monitoring of up to 10,000 peptides derived from a TMT-SH labeled proteome, real-time peptide identification by peak matching, and subsequent selection of endogenous, multiplexed single cell samples only when a carrier proteome peptide is identified. This technique improves proteomics depth in both MS2 and MS3 analyses for peptides on the user’s inclusion list. The second approach introduces a novel API programming platform, called inSeqAPI, that enables facile construction of real-time data analysis methods. We demonstrate its utility by constructing a method that performs a real-time search on a TMT-SH labeled carrier proteome and triggers quantitative MS2 or MS3 analysis on endogenous TMT labeled single cell proteomes only when a carrier proteome peptide is identified. This unbiased approach enables deep proteomic analysis and sensitive quantification of single cell proteomes by quantifying peptides only after they are identified and removing the carrier proteome from isolation during quantitative scans.