a Cartoon depicting the influence of ion sampling (injection time, IT) on single-cell signal. b Evaluation of the effect of increased ion sampling. Top: Histograms of the mean log2 signal-to-noise (s/n) values of all single-cell channel measurements per protein for all four methods. Bottom: Histograms of the mean Coefficient of Variation (CV) per protein for all four methods. Up to 14 CVs per protein were calculated by normalizing the three replicates of each method by equalizing the median s/n of the single-cell channels for each protein across replicates and dividing the standard deviation of the normalized s/n of each protein in each single-cell channel across replicates by the mean of the raw s/n for each protein in each single-cell channel across replicates. The mean CV of up to 14 CVs per protein was reported, as CV calculation was only performed for n = 3. c Density plot of protein log2 s/n values and their CV from all four methods (i.e. up to 14 CVs per protein per method). d Pearson correlation coefficient of fold changes between LSC and blast in single-cell samples and MS3-level bulk data (n=number of proteins). Left: For each method, only proteins without missing values were considered (i.e. proteins quantified in all 14 channels in each replicate). Right: Only proteins overlapping between all methods in left were considered. Fold changes in single-cell samples were calculated from the means of all LSCs (n = 15) and blast (n = 12). e Silhouette coefficients of LSC (n = 15) and blast (n = 12) for each method, calculated in PCA space using the same protein selection as in panel d. Boxplot shows median, 0.25 and 0.75 quantile, and whiskers extend to points within 1.5 interquartile range of lower and upper quartile. Source data are provided as a Source Data file.