Fig. 3: Quantitative performance characteristics of the micro-flow LC–MS/MS system.

a Design of a long-term performance test consisting of 10 cycles of 155 injections each. In each cycle, 20 replicates of 2 µg HeLa, 5 µg urine, 5 µg cerebrospinal fluid (CSF), and 5 µg each of 4 replicates of plasma digests from five individuals, as well as one deep-scale placenta digest (200 µg protein digest separted into 48 fractions) were analyzed using micro-flow LC–MS/MS using the stated gradient times. Between each sample type, 3 replicates of 500 fmol PROCAL synthetic retention time standards were injected. b Retention time stability of PROCAL peptides measured alone or spiked into body fluids (urine, CSF, plasma samples; main plot). The equation represents the linear model that was fitted to the data (R², squared Pearson correlation coefficient). The bottom right inset shows an expanded view of the main plot showing the retention time distribution of three closely eluting PROCAL peptides (in different colors) across all experiments. The top left inset summarizes the carry-over analysis across all 10 cycles (columns denote average carry-over and error bars denote the standard deviation). Source data are provided as a Source Data file. c Cumulative density plot showing the inter-cycle reproducibility of protein quantification for 200 injections of HeLa, urine and CSF, and 40 injections of five individual plasma samples (common proteins only). Dotted lines denotes the percentage of proteins that show <20% coefficient of variation (CV) in the analysis The inset shows a t‐SNE analysis of the 40 plasma injections measured from each of five individuals across the 10 cycles. d Same as in c but at the peptide level.