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Fig. 1 | Genome Biology

Fig. 1

From: Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2

Fig. 1

Optimizing and benchmarking MS analysis with bulk standards modeling SCoPE2 sets. a Conceptual diagram and work flow of SCoPE2. Cells are sorted into multiwell plates and lysed by mPOP [24]. The proteins in the lysates are digested with trypsin; the resulting peptides labeled with TMT, combined, and analyzed by LC-MS/MS. SCoPE2 sets contain reference channels that allow merging single cells from different SCoPE2 sets into a single dataset. The LC-MS/MS analysis is optimized by DO-MS [25], and peptide identification enhanced by DART-ID [26]. b Schematic for the design of a 100xM bulk standards. Monocytes (U937 cells) and embryonic kidney cells (HEK-293) were serially diluted to the indicated cell numbers, lysed, digested, and labeled with tandem-mass tags having the indicated reporter ions (RI). c Comparison of protein fold change between the embryonic kidney cells and monocytes estimated from the small samples and from the carrier samples of a 1xM standard, i.e., 1% sample from the 100xM standard described in a. The relative protein levels measured from bulk samples diluted to single-cell levels are very similar to the corresponding estimates from the isobaric carrier (bulk) samples. d Principal component analysis separates samples corresponding to embryonic kidney cells (HEK-293) or to monocytes (U-937 cells). The small samples (which correspond to bulk cell lysates diluted to single-cell level) cluster with the corresponding carrier samples, indicating that relative protein quantification from all samples is consistent and based on cell type. All quantified proteins were used for this analysis, and each protein was normalized separately for the carrier channels and the small sample channels

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