PNNL

Abstract

There is a growing demand to develop high throughput and high sensitivity mass spectrometry methods for single-cell proteomics. The commonly used isobaric labeling-based multiplexed single-cell proteomics approach suffered from distorted protein quantification due to co-isolated interfering ions during MS/MS fragmentation, known as ratio compression. We reasoned the use of MS3-based quantification could mitigate ratio compression and provide accurate quantification. However, previous studies indicated reduced proteome coverages in the MS3 method, likely due to long duty cycle time and ion losses during multi-level ion selection and fragmentation. Herein we described an improved MS acquisition method for MS3-based single-cell proteomics by employing a linear ion trap to measure reporter ions. We demonstrated linear ion trap can increase the proteome coverages for single-cell-level peptides and higher gains were obtained for MS3 methods. The optimized real-time search MS3 method was further applied to study the immune activation of single macrophages. Among the total of 168 single cells, over 1200 and 1000 proteins were quantifiable when 50% and 75% valid values were required, respectively. Our evaluation also revealed several limitations of the low-resolution ion trap detector for multiplexed single-cell proteomics and suggested experimental solutions to minimize their impacts on single-cell analysis.