Table 2.
A comparison of the modification percentage under several different digestion conditions for β-2-microglobulin, cytochrome c and myoglobin. All the experiments were repeated three times, and the means and standard deviations are reported.
| residue | overnight | 2 h | microwave | ultrasonic | 2+24 h | % SASA ratio1 |
|---|---|---|---|---|---|---|
| β-2-microglobulin2 | ||||||
| H13 | 10 ± 3 | 42.7 ± 0.7 | 14 ± 9 | 27.7 ± 0.3 | 10 ± 2 | 50.8 |
| H31 | 0.1 ± 0.13 | 7 ± 1 | N.D.4 | 2 ± 23 | N.D. | 32.7 |
| H51 | N.D. | 10 ± 1 | N.D. | N.D. | 0.7 ± 0.73 | 54.7 |
| S11 | 10 ± 5 | 8 ± 1 | 7 ± 3 | 5 ± 3 | 7 ± 3 | 11.4 |
| S20 | 1.1 ± 0.5 | 0.7 ± 0.1 | 0.8 ± 0.4 | 0.4 ± 0.3 | 0.7 ± 0.3 | 65.0 |
| S28 | 0.6 ± 0.2 | 0.31 ± 0.03 | N.D. | N.D. | N.D. | 19.7 |
| S33 | 2.4 ± 0.8 | 1.3 ± 0.3 | N.D. | 1 ± 13 | N.D. | 66.4 |
| S55 | 2 ± 23 | 3.4 ± 0.6 | N.D. | N.D. | N.D. | 55.9 |
| S57/K58 | 2 ± 23 | 3.3 ± 0.6 | N.D. | N.D. | N.D. | 35.2/96.3 |
| Y10 | N.D. | 0.065 ± 0.008 | N.D. | N.D. | N.D. | 41.9 |
| Y67/T68 | N.D. | 1.6 ± 0.8 | N.D. | N.D. | N.D. | 22.5/2.5 |
| Y78 | N.D. | 0.7 ± 0.3 | N.D. | N.D. | N.D. | 6.0 |
| N-terminus | 2.8 ± 0.4 | 2.4 ± 0.4 | 3 ± 2 | 2.3 ± 0.6 | 3.7 ± 0.7 | 85.0 |
| K6 | 0.20 ± 0.06 | 0.20 ± 0.07 | 0.2 ± 0.23 | 0.24 ± 0.05 | N.D. | 80.1 |
| K19 | 1.1 ± 0.3 | 4.1 ± 0.1 | 2 ± 1 | 3 ± 1 | 1.1 ± 0.2 | 69.0 |
| K41 | 2.3 ± 0.5 | 2.6 ± 0.7 | N.D. | N.D. | 1.9 ± 0.9 | 14.1 |
| K48 | 13 ± 2 | 8 ± 2 | N.D. | 13 ± 1 | 12 ± 2 | 81.8 |
| K75 | 0.6 ± 0.1 | 0.4 ± 0.2 | N.D. | N.D. | N.D. | 92.3 |
| K91 | 1.5 ± 0.3 | 1.4 ± 0.3 | 1 ± 13 | 1.0 ± 0.3 | 1.0 ± 0.2 | 61.5 |
| K94 | 2.3 ± 0.4 | 2.1 ± 0.5 | 2 ± 1 | 1.4 ± 0.4 | 1.5 ± 0.3 | 67.8 |
| % labeled5 | 76% | 95% | 38% | 52% | 48% | |
| cytochrome C2 | ||||||
| K25 | 30 ± 5 | 24 ± 2 | 13 ± 6 | 7 ± 4 | 23 ± 1 | 90.7 |
| K39 | 17 ± 2 | 20 ± 3 | 18 ± 4 | 19 ± 5 | 25 ± 3 | 72.9 |
| K53 | 2.6 ± 0.2 | 4.6 ± 0.5 | 3 ± 1 | 3.4 ± 0.9 | 1.1 ± 0.7 | 63.0 |
| K55 | 6 ± 1 | 9.1 ± 0.5 | 3 ± 33 | 7 ± 3 | 9.6 ± 0.7 | 36.5 |
| K60 | 1.6 ± 0.1 | 1.60 ± 0.08 | N.D. | N.D. | N.D. | 50.0 |
| K72 | 60 ± 3 | 56 ± 3 | N.D. | N.D. | 60 ± 10 | 75.3 |
| K73 | 18 ± 2 | 27 ± 1 | N.D. | N.D. | 17 ± 7 | 62.6 |
| K86 | 7 ± 1 | 10 ± 1 | 7 ± 2 | 8 ± 1 | 1 ± 13 | 41.6 |
| K87 | 9.3 ± 0.6 | 9.5 ± 0.9 | 8 ± 1 | 10.0 ± 0.3 | 8.1 ± 0.6 | 86.8 |
| K88 | 1.6 ± 0.1 | 1.7 ± 0.2 | 1.4 ± 0.2 | 1.77 ± 0.04 | 1.4 ± 0.1 | 94.0 |
| K99 | 45 ± 3 | 40 ± 10 | 30 ± 10 | 24 ± 2 | 14 ± 6 | 44.2 |
| K100 | 24 ± 2 | 22 ± 3 | 14 ± 6 | 14 ± 2 | 30 ± 10 | 50.4 |
| % labeled5 | 45% | 45% | 33% | 33% | 41% | |
| Myoglobin2 | ||||||
| H24 | N.D. | 3.1 ± 0.4 | N.D. | N.D. | N.D. | 2.7 |
| H36 | 1.9 ± 0.7 | 6.4 ± 0.5 | 2.9 ± 0.9 | 1.0 ± 0.1 | 3.2 ± 0.5 | 32.6 |
| H81 | 9 ± 5 | 38 ± 1 | 18 ± 2 | 9 ± 1 | 1 ± 13 | 89.1 |
| H116 | 11 ± 7 | 33.1 ± 0.7 | N.D. | N.D. | N.D. | 43.9 |
| S117 | 0.6 ± 0.4 | 1.74 ± 0.04 | N.D. | N.D. | N.D. | 51.3 |
| N-terminus | 11 ± 1 | 16 ± 3 | 19 ± 1 | 13 ± 2 | 39 ± 2 | 97.8 |
| K16 | 1.8 ± 0.7 | 1.3 ± 0.8 | 1 ± 23 | 2.3 ± 0.3 | 0.8 ± 0.83 | 22.1 |
| K42 | 0.24 ± 0.08 | 0.81 ± 0.06 | 0.4 ± 0.1 | 0.12 ± 0.02 | 0.40 ± 0.07 | 33.0 |
| K45 | 0.24 ± 0.08 | 0.81 ± 0.06 | 0.4 ± 0.1 | 0.12 ± 0.02 | 0.40 ± 0.07 | 54.9 |
| K77 | 19 ± 6 | 42 ± 8 | 45 ± 8 | 19 ± 3 | 3 ± 53 | 50.5 |
| K78 | 36 ± 6 | 37 ± 4 | 44 ± 6 | 27 ± 8 | 2 ± 23 | 46.2 |
| K79 | 6 ± 3 | 13 ± 2 | 14 ± 3 | 6 ± 3 | 1 ± 13 | 53.6 |
| % labeled5 | 35% | 39% | 29% | 29% | 29% | |
SASA was calculated using GETAREA 1.1 [45]. 1.4 Å was used as the probe radius, and the calculated SASA percentage is the ratio of the SASA of the side chain in the protein to the SASA of the side chain (X) in the unstructured Gly-X-Gly tripeptide. Residues with %SASA values that exceed 50% are typically considered to be solvent exposed, and residues with ratios less than 20% are typically considered to be buried. We chose 30% as the cutoff for determining if a residue is solvent exposed.
The PDB IDs for β-2-microglobulin, cytochrome c and myoglobin that were used to determine SASA were 1JNJ, 1AKK and 1DWR, respectively. For β-2-microglobulin, 1JNJ consists of 20 NMR structures, and so the reported SASA values are the average from these 20 structures.
This modified peptide is not detected in every experiment.
N.D. indicates that modified peptide is not detected in any experiment under these conditions.
% labeled corresponds to the percentage of the surface exposed modifiable (i.e. His, Lys, Cys, Ser, Thr, and Tyr) residues that are found to be labeled. A complete list of modifiable residues in β-2-microglobulin, cytochrome c and myoglobin and their calculated SASA values can be found in Table S1 in the Supplemental Information.