Fig. 1: Optimization of sample preparation by surface maximization and reduction of dimensionality (SMRD).

a Visualization of the pre-analytical sample preparation workflow and protein extraction; FFPE: formalin-fixed paraffin-embedded; LC-MS/MS: liquid chromatography-coupled tandem mass spectrometry; TMT: tandem mass tags. b Exemplary micrographs of input tissue (upper) and the resulting fragments (lower image) from the experiments in subplots d–h. c TMT-LC-MS/MS analysis of the standard protocol (Ctrl, lighter colors) and three technical replicates of the optimized protocol; protein intensities were normalized to a second control; relative abundances of membrane (red) and non-membrane proteins (blue) are shown as boxplots with median and inter-quartile range (IQR), whiskers extend to 1.5 IQR of lower and upper quartile respectively. d Fraction of non-tryptic peptides when using either a highly specific collagenase (red) or a commercial enzyme (mix) (dark red) for single-cell isolation compared to omission of any enzymatic matrix digestion (blue); bars are means, whiskers are standard deviations, dots are single data points (applies to all subplots d–h); NSCLC n = 2/2/1 technically independent samples; UCC n = 3/3/1. e Legend and number of replicates (by sample type) for subplots f–h; UCC#1/#2/#3/#4/NSCLC; Opt.: optimized; DG: deglycosylated; com.: commercial enzyme (mix); f Number of proteins identified in a single MS run without fractionation and without matching spectra between runs; please see methods for the different technical setups; g Number of membrane proteins; h Fraction of membrane proteins. Source data are provided as a Source Data file.