ANALYSIS OF THE LIPIDATED RECOMBINANT OUTER SURFACE PROTEIN-A FROM BORRELIA-BURGDORFERI BY MASS-SPECTROMETRY

The outer surface protein A, OspA, from the spirochete Borrelia burgdo rferi is a lipoprotein of 25 kDa. The recombinant OspA (rOspA) express ed in Escherichia coli has been purified and analyzed by electro-spray mass spectrometry (ESMS). A heterogenous spectrum gave a measured mas s of 28,462 +/- 9 Da for the major component compared to an expected m ass of 28,445 Da (Delta m = +17 Da), and a measured mass of 28,228 +/- 7 Pa for a minor component. The theoretical mass is based on the N-te rminal being an s(palmitoyloxy)-(2R,S)-propyl]-N-palmitoylcysteine mod ification according to the model established by Hantke and Braun (Eur. J. Biochem. 34, 284-296, 1973) for bacterial lipoproteins. To determi ne whether rOspA conforms to this model, a complementary detailed anal ysis of this lipidation was necessary. The fatty acid content of the c omplete protein as analyzed by gas chromatography-mass spectrometry re vealed saturated fatty acids ranging from C14 to C18 as well as C16 an d C18 unsaturated fatty acids, with palmitate (C16:0) being the major component. Focusing on the lipid moieties, the N-terminal tryptic pept ide was purified by normal phase HPLC using a silica column and a grad ient of hexane in isopropanol, Analysis of the N-terminal peptide by E SMS and fast atom bombardment-mass spectrometry revealed a minor fract ion of rOspA molecules which contained only two C16 residues and that in addition to partial oxidation, the major N-terminal peptide had a m ass difference of -2 Da compared to a theoretical structure with three palmitate residues, indicating that one of the three fatty acid resid ues was unsaturated. Minor forms with mass differences of 28 Da were a lso observed, indicating that one of the three acyl residues was C14 i n one case and C18 in the other, instead of C16 in the major form. Ana lysis of the rOspA peptide backbone revealed that the sole methionine at position 22 was partially oxidized to a methionine sulfoxide. Thus the mass analysis of the major mass is consistent with a mixed populat ion of lipoprotein molecules containing variations not only in the lip id moiety contributing to an elevation in the mass of Delta m = 7 Da c ompared to the theoretical structure proposed, but also in the peptide chain. Partial oxidations at two points in the protein backbone (<30% of the population in each case) contribute to an additional augmentat ion in mass and thus can account for the remaining mass difference in the measured mass. (C) 1997 Academic Press.