The outer surface protein A, OspA, from the spirochete Borrelia burgdorferi is a lipoprotein of 25 kDa. The recombinant OspA (rOspA) expressed in Escherichia coli has been purified and analyzed by electrospray mass spectrometry (ESMS). A heterogenous spectrum gave a measured mass of 28,462 +/- 9 Da for the major component compared to an expected mass of 28,445 Da (Deltam = +17 Da), and a measured mass of 28,228 +/- 7 Da for a minor component. The theoretical mass is based on the N-terminal being an S-[2,3-bis(palmitoyloxy)-(2R, S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-N-palmitoylcysteine modification according to the model established by Hantke and Braun (Eur. J. Biochem. 34, 284-296, 1973) for bacterial lipoproteins. To determine whether rOspA conforms to this model, a complementary detailed analysis of this lipidation was necessary. The fatty acid content of the complete protein as analyzed by gas chromatography-mass spectrometry revealed saturated fatty acids ranging from C14 to C18 as well as C16 and C18 unsaturated fatty acids, with palmitate (C16:0) being the major component. Focusing on the lipid moieties, the N-terminal tryptic peptide was purified by normal phase HPLC using a silica column and a gradient of hexane in isopropanol. Analysis of the N-terminal peptide by ESMS and fast atom bombardment-mass spectrometry revealed a minor fraction of rOspA molecules which contained only two C16 residues and that in addition to partial oxidation, the major N-terminal peptide had a mass difference of -2 Da compared to a theoretical structure with three palmitate residues, indicating that one of the three fatty acid residues was unsaturated. Minor forms with mass differences of 28 Da were also observed, indicating that one of the three acyl residues was C14 in one case and C18 in the other, instead of C16 in the major form. Analysis 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 population of lipoprotein molecules containing variations not only in the lipid moiety contributing to an elevation in the mass of Deltam = 7 Da compared 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 augmentation in mass and thus can account for the remaining mass difference in the measured mass.