Automated sample preparation for high-throughput single-cell proteomics | Harrison Specht | SCP2018

Presentation by Harrison Specht at the first single-cell proteomics conference http://single-cell.net https://single-cell.net/proteomics/sc... | Preprint: https://doi.org/10.1101/399774
Data: https://scp.slavovlab.net/mPOP


A major limitation to applying quantitative LC-MS/MS proteomics to small samples, such as single cells, are the losses incurred during sample cleanup. To relieve this limitation, we developed a Minimal ProteOmic sample Preparation (mPOP) method for culture-grown mammalian cells. mPOP obviates cleanup and thus eliminates cleanup-related losses while expediting sample preparation and simplifying its automation. Bulk SILAC samples processed by mPOP or by conventional urea-based methods indicated that mPOP results in complete cell lysis and accurate relative quantification. We integrated mPOP lysis with the Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) sample preparation, and benchmarked the quantification of such samples on a Q-exactive instrument. The results demonstrate low noise and high technical reproducibility. Then, we FACS sorted single monocytes, macrophages and mouse ES cells into 386-well plates and analyzed them by automated mPOP and SCoPE-MS. This allowed us to prepare hundreds of SCoPE-MS samples per day and to analyze 20 samples (equivalent to 160 single cells) per day per instrument. We quantified thousands of proteins with multiple peptides per protein and reproducible relative quantification, CV = 15%. The quantified proteins enabled separating the single cells by cell-type and cell-division-cycle phase, and even identifying new subpopulations of macrophages.