Fig. 1: Qualitative performance characteristics of the micro-flow LC–MS/MS system.

a Comparison of the cross-sectional areas of LC columns of different inner diameters (ID). b Boxplots summarizing the chromatographic peak width distributions (full width at half-maximum, FWHM) of all identified peptides for different LC gradient times (2 µg HeLa protein digest injected). Boxes and whiskers cover 50% and 1.5 times the interquartile range of the data respectively. Numbers above boxes denote the median FWHM values (in seconds), numbers below boxes represent the number of peptides contained in the analysis. c Example base peak chromatogram of 2 µg HeLa protein digest separated by a 60 min LC gradient. Selected chromatographic peaks are labeled with the m/z and FWHM values of the underlying peptide. d Bar charts comparing peptide identification results obtained for different sample loadings and LC gradient times using either nano-flow^{29, 31} (red) or micro-flow LC–MS/MS (blue). White numbers inside bars denote the number of peptides. e Same as panel d but for proteins. f Box plots comparing Andromeda peptide identification scores obtained for different sample loadings and LC gradient times using either nano-flow (red, n = 24,113 for 30 min, 36,442 for 60 min) or micro-flow (blue, n = 19,514 (1 µg), 23,047 (5 µg), and 23,630 (10 µg) for 30 min, and n = 14,992 (1 µg), 34,356 (5 µg), and 37,130 (10 µg) for 60 min) LC–MS/MS. g Same as panel f but for peptide chromatographic peak widths. The number of peaks used for each box are 22,254 (nano), 19,104 (micro, 1 µg), 21,820 (micro, 5 µg), and 22,065 (micro, 10 µg) for 30 min, and 33,772 (nano), 12,831 (micro, 1 µg), 32,835 (micro, 5 µg), and 34,884 (micro, 10 µg) for 60 min. Boxes and whiskers are defined as in b. Source data are provided as a Source Data file for b, f, and g.