A sensitive and simple scheme was developed for the rapid separation of mitochondrial complex III subunits by reverse-phase high-performance liquid chromatography (reverse-phase HPLC). Ten of the 11 subunits of cytochrome bc1 complex were separated with nearly baseline resolution between each peak. Cytochrome b was precipitated by acetonitrile on the column and could not be analyzed; the 10 other polypeptides were positively identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrospray ionization mass spectrometry (ESI/MS). The ESI/MS-determined molecular masses for subunits II, VI, VIII, IX, and XI are in excellent agreement with previously reported values; i.e., all are within ±2 mass units per 10 kDa. None of the other subunits gave molecular masses that agree with the published sequence values. The molecular mass of subunit I is 49 236 Da, which is far greater than the molecular mass of 35 833 Da calculated from the reported DNA sequence [Gencic et al. (1991) Eur. J. Biochem. 199, 122–131]. The Fe-S protein (subunit V) gives two masses which differ by 60 mass units, presumably due to either the partial loss of the two sulfur atoms or microheterogeneity. Neither mass agrees with the sequence value, the larger mass being 39 mass units lower than expected from the sequence. The molecular masses of subunits VII and X are 81 and 129 Da larger, respectively, than those calculated from their sequences [Borchart et al. (1986) FEBS Lett. 200, 81–86; Schägger et al. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 307–311]. Moreover, the HPLC-purified subunit X contains two distinct components: 7298 (Xa) and 7326 Da (Xb), respectively. Initial sequence analysis of the tryptic digests of the two proteins by HPLC/ESI/MS/MS reveals that Xb contains the NH2-terminal sequence reported by Schagger et al. (1983), while in Xa alanine replaces the NH2-terminal valine. The data indicate the high precision of electrospray mass spectrometry in determining protein molecular masses and demonstrate its usefulness in verifying protein sequences or determining errors in them.
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