Dataset Information

Enhancing single-cell proteomics through tailored Data-Independent Acquisition and micropillar array-based chromatography

ABSTRACT: Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. However, the limited amount of material encapsulated in a single cell raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, mass spectrometry-based single-cell proteomics (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, albeit further development is needed to realize its full potential. To this end, we carried out comprehensive analysis of orbitrap-based data independent acquisition (DIA) for limited material proteomics. Notably, we found a fundamental difference between optimal DIA methods for high- and low-load samples. We further improved our low-input DIA method by relying on high-resolution MS1 quantification, thus more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we were able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we further enhanced our workflow by combining DIA with the latest chromatographic and computational advances and concluded with showcasing our developed workflow on profiling real single-cell proteomes. THE INCLUDED README FILE CONTAINS THE ASSOCIATION BETWEEN THE MANUSCRIPT FIGURES AND RAW FILES

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ertwin M. Schoof