Table 1.
Overview of LC methods developed and tested in this study.
| Method | Method overviewa | Peptide loaded [μg] | Runtime [h]b |
|---|---|---|---|
| 2D-LC (all with 50 cm analytical column) | |||
| 2D|3salt4.5 | bt; 5, 100, 2000 mM (each 300 min) | 4.5 | 20 |
| 2D|3salt9 | bt; 5, 100, 2000 mM (each 300 min) | 9 | 20 |
| 2D|11salt4.5 | bt; 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000 mM (each 120 min) | 4.5 | 24 |
| 2D|11salt9 | bt; 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000 mM (each 120 min) | 9 | 24 |
| 2D|10pH_G1 | bt; pH 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0 (each 120 min) | 4.5 | 22 |
| 2D|10pH_G24.5 | bt (300 min); pH 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0 (each 120 min) | 4.5 | 25 |
| 2D|10pH_G29 | bt (300 min); pH 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0 (each 120 min) | 9 | 25 |
| 2D|5pH+2salt | bt (228 min); 1 mM (228 min); pH 3.0 (120 min), 4.0 (105 min), 4.5 (90 min), 5.0 (90 min), 6.0 (79 min); 2000 mM (60 min) | 4.5 | 16.7 |
| 2D|6pH+1salt_G1 | bt (228 min); pH 2.5 (228 min), 3.5 (120 min), 4.5 (90 min), 5.0 (90 min), 6.0 (79 min), 8.0 (60 min); 2000 mM (60 min) | 4.5 | 15.9 |
| 2D|6pH+1salt_G2 | bt (466 min); pH 2.5 (228 min), 3.5 (120 min), 4.5 (90 min), 5.0 (90 min), 6.0 (79 min), 8.0 (60 min); 2000 mM (60 min) | 4.5 | 19.9 |
| 2D|8pH+1salt_G14.5 | bt (300 min); pH 2.5 (120 min), 3.0 (120 min), 3.5 (120 min), 4.0 (105 min), 4.5 (105 min), 5.0 (105 min), 6.0 (105 min), 8.0 (80 min); 2000 mM (80 min) | 4.5 | 20.7 |
| 2D|8pH+1salt_G19 | bt (300 min); pH 2.5 (120 min), 3.0 (120 min), 3.5 (120 min), 4.0 (105 min), 4.5 (105 min), 5.0 (105 min), 6.0 (105 min), 8.0 (80 min); 2000 mM (80 min) | 9 | 20.7 |
| 2D|8pH+1salt_G2 | bt (228 min); pH 2.5 (228 min), 3.0 (120 min), 3.5 (120 min), 4.0 (105 min), 4.5 (90 min), 5.0 (90 min), 6.0 (79 min), 8.0 (60 min); 2000 mM (60 min) | 4.5 | 19.7 |
| 2D|8pH+4salt4.5 | bt (300 min); 1 mM (120 min); pH 2.5 (120 min), 3.0 (120 min), 3.5 (120 min), 4.0 (105 min), 4.5 (105 min), 5.0 (105 min), 6.0 (105 min), 8.0 (80 min); 500 mM (80 min), 1000 mM (80 min), 2000 mM (80 min) | 4.5 | 25.3 |
| 2D|8pH+4salt9 | bt (300 min); 1 mM (120 min); pH 2.5 (120 min), 3.0 (120 min), 3.5 (120 min), 4.0 (105 min), 4.5 (105 min), 5.0 (105 min), 6.0 (105 min), 8.0 (80 min); 500 mM (80 min); 1000 mM (80 min); 2000 mM (80 min) | 9 | 25.3 |
| 2D|10pH+1salt | bt (228 min); pH 2.5 (228 min), 3.0 (120 min), 3.5 (120 min), 4.0 (105 min), 4.5 (90 min), 5.0 (90 min), 5.5 (90 min), 6.0 (79 min), 7.0 (79 min), 8.0 (60 min), 2000 mM (60 min) | 4.5 | 22.5 |
| 2D|11saltc | bt; 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000 mM (each 120 min) | 11 | 24 |
| 1D-LC | |||
| 50 cm analytical column | |||
| 1D|8h_501.6 | 460min_Pre20 (460 min plus 20 min pre-equilibration) | 1.6 | 8 |
| 1D|8h_502 | 460min_Pre20 (460 min plus 20 min pre-equilibration) | 2 | 8 |
| 1D|8h_502.5 | 460min_Pre30 (460 min plus 30 min pre-equilibration to also work with the 75 cm column) | 2.5 | 8.2 |
| 1D|12h_50 | 720min_Pre30 (720 min plus 30 min pre-equilibration to also work with the 75 cm column) | 2.5 | 12.5 |
| 1D|4hc | 260min_Pre20 (260 min plus 20 min pre-equilibration) | 0.8–2 | 4.7 |
| 1D|8hc | 460min_Pre20 (460 min plus 20 min pre-equilibration) | 2 | 8 |
| 75 cm analytical column | |||
| 1D|8h_75 | 460min_Pre30 (460 min plus 30 min pre-equilibration) | 2.5 | 8.2 |
| 1D|12h_75 | 720min_Pre30 (720 min plus 30 min pre-equilibration) | 2.5 | 12.5 |
| GeLC (50 cm analytical column) | |||
| GeLC | 10x (Load, 90 min) | (30–60)d | 16.7 |
a For description of the gradients, see Supplementary Table S1. bt, breakthrough; i.e., peptides that do not sufficiently bind to the SCX column and are directly eluted onto the C18 RP column before any salt or pH steps. Concentrations correspond to NaCl concentrations in loading buffer B. pH values correspond to commercial pH plugs (CTIBiphase buffers, Column Technology, Inc.). bMethod runtime without intermediary times for washing etc., but with pre-equilibration for 1D methods. cThis method was run on four biological replicates as described by Kleiner et al. (2017). dProtein concentration was determined before loading samples on the gel.