Figure 4.

RT shifts induced by different amounts of protein loaded on the column can be compensated using on-the-fly RT calibration. Grey lines represent peptide retention times when varying the sample load on the column. Black crosses indicate peptides that would have been missed without using on-the-fly RT calibration. With 2μg protein loaded on the column 111, 122, and 49 peptides would elute outside the retention time window and hence be missed in the three technical replicates. The earliest eluting peptide was missed once even using on-the-fly RT calibration in the second run of the 1μg load.