SUBUNIT ANALYSIS OF BOVINE CYTOCHROME BC1 BY REVERSE-PHASE HPLC AND DETERMINATION OF THE SUBUNIT MOLECULAR MASSES BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

A sensitive and simple scheme was developed for the rapid separation o f mitochondrial complex III subunits by reverse-phase high-performance liquid chromatography (reverse-phase HPLC). Ten of the 11 subunits of cytochrome bc(1) complex were separated with nearly baseline resoluti on 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 ele ctrophoresis (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 v alues; i.e., all are within +/-2 mass units per 10 kDa. None of the ot her subunits gave molecular masses that agree with the published seque nce 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 repo rted 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 mas s units, presumably due to either the partial loss of the two sulfur a toms or microheterogeneity. Neither mass agrees with the sequence valu e, the larger mass being 39 mass units lower than expected from the se quence. The molecular masses of subunits VII and X are 81 and 129 Da l arger, respectively, than those calculated from their sequences [Borch art et al. (1986) FEBS Lett. 200, 81-86; Schagger 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 th e two proteins by HPLC/ESI/MS/MS reveals that Xb contains the NH2-term inal 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 ma sses and demonstrate its usefulness in verifying protein sequences or determining errors in them.