TABLE 1.
MALDI-TOF analysis of NEM-alkylated, Cys-containing peptides from the bovine and rat complex I 75-kDa subunit Experimental masses were obtained from isolated and denatured complex I (2 mg/ml) treated with DTT (10 mm) and SDS (1%) for 10 min at 37°C; the DTT was removed; and the samples were treated with NEM. Bovine or rat mitochondria (1 mg of protein/ml) were incubated with diamide (0.5 mm) for 5 min at 37°C. After incubation all samples were alkylated with NEM (25–50 mm) and separated by electrophoresis (one-dimensional SDS-PAGE for isolated complex I samples and two-dimensional BN/SDS-PAGE for mitochondria) before in-gel digestion and MALDI-TOF.
|
Cys
no.a |
Sequenceb |
Enzymec |
FeSd |
Mass of NEM-alkylated peptide (MH+) |
|||
|---|---|---|---|---|---|---|---|
|
Calculatede |
Observed |
||||||
| Denatured complex I | Bovine mitochondria (b) | Rat mitochondria (r) | |||||
| 30 | TATAASNLIEVFVDGQSVMVEPGTTVLQACEK (b) | T | No | 3433.6865 | 3433.6977 | 3433.6301 | |
| TGTAASNLIEVFVDGQSVMVEPGTTVLQACEK (r) | 3419.6707 | 3419.7310 | |||||
| 41 | FCYHER | T | Yes | 979.4090 | 979.4006 | 979.4030 | 979.4123 |
| 52 | LSVAGNCR | T | Yes | 944.4618 | 944.4646 | 944.4555 | 944.4660 |
| 55 | MCLVEIEK | T | Yes | 1089.5319 | 1089.5171 | 1089.5236 | 1089.5527 |
| 69 | VVAACAMPVMK | T | Yes | 1244.6200 | 1244.6146 | 1244.6105 | 1244.6212 |
| 105,108 | DCPIC or DCPIC | D | Yes | 675.2475 | |||
| DCPIC | 800.2945 | ||||||
| 114 | DQGGEC | D | Yes | 733.2456 | |||
| 153,156 | CIQCTR or CIQCTR | T | Yes | 848.3753 | 848.3709 | 848.3825 | 848.3458 |
| CIQCTR | 973.4230 | 973.4194 | 973.4202 | 973.4334 | |||
| 159 | CIR | T | Yes | 516.2597 | |||
| 203 | DICPVGALTSKPYAFTARPW | D | Yes | 2318.1564 | 2318.1790 | 2318.2058 | 2318.2073 |
| 344 | VDSDTLCTEEVFPTAGAGTDLR (b) | T | No | 2422.1078 | 2422.0918 | 2422.0857 | |
| VDSDTLCTEEIFPNEGAGTDLR (r) | 2507.1241 | 2507.1228 | |||||
| 531 | MLFLLGADGGCITR (b) | T | No | 1591.7970 | 1591.7957 | 1591.7982 | |
| LLFLLGADGGCITR (r) | 1573.8405 | 1573.8358 | |||||
| 541 | DCFIVYQGHHG | D | No | 1400.5979 | 1400.5907 | 1400.6039 | 1400.6027 |
| 687 | DSISRASQTMAKCVKAVTEGAHAVE (b) | D | No | 2714.3163 | 2714.3508 | 2714.3940 | |
| DSISRASQTMAKCVKAVTEGAQAVE (r) | 2705.3231 | 2705.3904 | |||||
| 704 | AVTEGAHAVEEPSIC (b) | T | No | 1637.7475 | 1637.7476 | 1637.7484 | |
| AVTEGAQAVEEPSIC (r) (–NEM) | 1503.6995 | 1503.6367 | |||||
Cysteine residue numbers are shown, which are the same for bovine and rat sequences
Unless specified as bovine (b) or rat (r), peptide sequences are the same for both species
T indicates trypsin, and D indicates Asp-N
Data indicate whether the cysteine residue is part of an iron-sulfur cluster
Calculated masses of peptides were alkylated with NEM (+125.047 Da)