Supplementary Figure 2: Determination of retention times and retention time indices

(a) Retention time stability of 71 selected retention time standard peptides (66 RT peptides + 5 QC peptides) across ~1,200 LC-MS/MS runs (identification in n runs and median indicated). The median standard deviation of observed retention time differences for individual peptides was 48.7 seconds without (middle panel) and 12.4 seconds with retention time adjustment (lower panel) using RT indices calculated based on the peptides ISLGEHEGGGK and YDTAIDFGLFK. Data were filtered for Andromeda scores of >100. (b) The scatter plot of two retention time indices calculated based on different reference peptides shows that a conversion between them is possible without losing accuracy (R² = 0.9996). Retention time index 1 (iRT1) is calculated based on the early eluting peptide ISLGEHEGGGK and late eluting peptide YDTAIDFGLFK (same as in (a); indicated by purple dashed lines). Retention time index 2 (iRT2) is calculated based on SYASDFGSSAK and GFVIDDGLITK (red dashed line). Each dot represents one of the 71 selected peptides identified in one of the ~1200 LC-MS/MS runs. (c) Similar to (b), here a third retention time index (iRT3) was calculated based on GSGGFTEFDLK and LTDELLSEYYK (orange dashed line) which span only a narrow part of the gradient. The linear fit shows that retention time indices can still be converted with very high accuracy (R² = 0.9977) indicating that any high confident identifications (not necessarily peptides used for retention time calculation) can be used for retention time index calculation and thus conversion.