Table I.
Disulfide Bond Scrambling Dependence on Cysteine Content and Experimental Conditions
| Protein(s), experiment | Cys (%) | No.of expts | Valid crosslinks | Types (%) | False-positive crosslinks | Types (%) | Scrambling (%) | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 0 | 1 | 2 | ||||||
| αB-crystallin | 0 | 3 | 28 | 32 | 11 | 57 | 0 | 0 | 0 | 0 | 0 |
| Hsp21, short trypsin | 0.5 | 1 | 42 | 31 | 14 | 55 | 0 | 0 | 0 | 0 | 0 |
| αB-crystallin + C8/α7 | 0.9 | 4 | 51 | 46 | 10 | 42 | 1 | 0 | 0 | 2 | 2 |
| Hsp21 | 0.5 | 8 | 42 | 37 | 5 | 56 | 1 | 0 | 0 | 2 | 2 |
| Hsp21 + CS | 0.8 | 3 | 38 | 28 | 13 | 56 | 1 | 0 | 0 | 3 | 3 |
| MDH, short trypsin | 2.5 | 1 | 15 | 48 | 14 | 10 | 6 | 0 | 5 | 24 | 29 |
| Hsp21 + MDH | 1.8 | 1 | 27 | 37 | 11 | 24 | 11 | 8 | 3 | 18 | 29 |
| CS | 0.9 | 4 | 11 | 39 | 6 | 17 | 7 | 0 | 6 | 33 | 39 |
| MDH | 2.5 | 4 | 2 | 0 | 6 | 0 | 29 | 48 | 6 | 39 | 94 |
Proteins were crosslinked and analyzed according to the workflow described in Figure 1, or with a modification in the experiment as indicated in the left-most column: short trypsin = digestion for 1 hr at a protease:protein ration of 1:10 instead of 1:100. Crosslinks were designated “valid” or “false-positive,” depending on their isotope pattern. If the peak at +4 Da was lower than the “light” and “heavy” peaks (X < 1), the crosslink was considered valid; if it was higher than the “light” and “heavy” peaks (X > 1), it was considered a false-positive. Crosslink type 0 indicates a dead-end, 1 an intrapeptide, and 2 an interpeptide crosslink. The scrambling percentage is the percentage of false-positive crosslinks of the total (valid and false-positive crosslinks).