Automated single-cell proteomics providing sufficient proteome depth to study complex biology beyond cell type classifications

Abstract

Mass spectrometry (MS)-based single-cell proteomics (SCP) has gained massive attention as a viable complement to other single cell approaches. The rapid technological and computational advances in the field have pushed the boundaries of sensitivity and throughput. However, reproducible quantification of thousands of proteins within a single cell at reasonable proteome depth to characterize biological phenomena remains a challenge. To address some of those limitations we present a combination of fully automated single cell sample preparation utilizing a dedicated chip within the picolitre dispensing robot, the cellenONE. The proteoCHIP EVO 96 can be directly interfaced with the Evosep One chromatographic system for in-line desalting and highly reproducible separation with a throughput of 80 samples per day. This, in combination with the Bruker timsTOF MS instruments, demonstrates double the identifications without manual sample handling. Moreover, relative to standard high-performance liquid chromatography, the Evosep One separation provides further 2-fold improvement in protein identifications. The implementation of the newest generation timsTOF Ultra with our proteoCHIP EVO 96-based sample preparation workflow reproducibly identifies up to 4,000 proteins per single HEK-293T without a carrier or match-between runs. Our current SCP depth spans over 4 orders of magnitude and identifies over 50 biologically relevant ubiquitin ligases. We complement our highly reproducible single-cell proteomics workflow to profile hundreds of lipopolysaccharide (LPS)-perturbed THP-1 cells and identified key regulatory proteins involved in interleukin and interferon signaling. This study demonstrates that the proteoCHIP EVO 96-based SCP sample preparation with the timsTOF Ultra provides sufficient proteome depth to study complex biology beyond cell-type classifications.

Evaluation of Protein Identification and Quantification by the diaPASEF Method on timsTOF SCP

“…The modification resulted in a remarkable 19-fold increase in sensitivity at the single-cell-equivalent level, allowing the identification of 3888 proteins with 1 ng of HeLa peptides. The detection sensitivity can be further improved using the newly released timsTOF Ultra instrument …”

Instrumentation at the Leading Edge of Proteomics

“…Since more ions can be stored and manipulated in the v.4 TIMS cartridge, a wider dynamic range of 1 × 10 5 is seen in the HT and Flex relative to 5 × 10 4 in the SCP. − The Flex is compatible with both ESI and matrix-assisted laser desorption/ionization (MALDI) ion sources, , and real-time data analysis through the parallel search engine in real-time (PaSER) is available on the timsTOF Pro 2 and other newer models. , The recently released timsTOF Ultra enables scan speeds approaching 300 Hz by integrating the sensitivity of the timsTOF SCP with the dynamic range performance of the timsTOF HT, all while creating superior front-end ion transfer efficiency compatible with high scan speeds − Although not commercialized, UVPD and ECD have been coupled to timsTOF instruments with varying degrees of in-house modifications − …”

Cheap, Robust and Versatile Two-Dimensional Chromatography system for Proteomics of Nanogram Scale Samples

“…The combination of these two analytical tools on the analysis of peptides from cells or tissues has become the state-of-the art workflow in proteomics. New mass spectrometry configurations 8, 9 combined with robust nanoLC 10, 11 has led to considerable increase in proteome coverage and throughput. Nevertheless, deep and comprehensive proteome characterization by single-shot LC-MS still represents a challenge.…”