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Fig. 3 | Genome Biology

Fig. 3

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

Fig. 3

Model system and technical benchmarks for analyzed single cells and proteins. a Monocytes were differentiated into macrophages by PMA treatment, and FACS-sorted cells prepared into 179 SCoPE2 sets, labeled with TMT 11-plex or TMT 16-plex. b Distributions of coefficients of variation (CVs) for the fold changes of peptides originating from the same protein. The CVs for single cells are significantly lower than for the control wells. c A distribution of time differences between the apex of chromatographic peaks and the time when they were sampled for MS2 analysis, see Box 1. Over 80% of ions (shaded box) were sampled within 3 s of their apexes. d A distribution of precursor ion fractions (PIF) for all peptides across all SCoPE2 sets. PIF is a quantitative metric computed by MaxQuant [33] to estimate the degree of coisolation. For most MS2 scans, over 97% of the ions isolated for fragmentation and MS2 analysis belonged to a single precursor (peptide sequence), see Fig. 2. The square and the cross mark, the median, and the mean respectively. e About 35% of MS2 spectra are assigned to peptide sequences at 1% false discovery rate (FDR). The lower mode of the distribution corresponds to samples analyzed when the quadrupole of our instrument had suboptimal ion transmission performance. f The number of identified and quantified peptides and proteins in single cells from SCoPE2 sets analyzed on 60 min nLC gradients. All peptide and protein identifications are at FDR below 1% and are supported by DART-ID [26]. The criteria for stringent filtering are described in the “Methods” section. See Additional file 1: Fig. S3b for the number of peptides and proteins identified from MS spectra alone. The number of proteins with non-zero RI intensity in control wells ranged from 66 to a few hundred in contaminated or cross-labeled control wells that were excluded from the analysis. The x-axes in all violin plots (vertical histograms) correspond to counts, number of MS2 scans in c, number of PSMs in d, and number of SCoPE2 sets in e and f

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