Plasmids were constructed for overexpression of the Escherichia coli dihydrolipoamide acetyltransferase (I-lip E2, with a single hybrid lipoyl domain per subunit) and dihydrolipoamide dehydrogenase (E3). A purification protocol is presented that yields homogeneous recombinant I-lip E2 and E3 proteins. The hybrid lipoyl domain was also expressed independently. Masses of 45,953 +/- 73Da (1-lip E2), 50,528 +/- 5.5 Da (apo-E3), 51,266 +/- 48 Da (E3 including FAD), and 8982 +/- 4.0 (lipoyl domain) were determined by MALDI-TOF mass spectrometry. The purified I-lip E2 and E3 proteins were functionally active according to the overall PDHc activity measurement. The lipoyl domain was fully acetylated after just 30 s of incubation with E I and pyruvate. The mass of the acetylated lipoyl domain is 9019 +/- 2Da according to MALDI-TOF mass spectrometry. Treatment of the I-lip E2 subunit with trypsin resulted in the appearance of the lipoyl domain with a mass of 10,112 +/- 3 Da. When preincubated with E1 and pyruvate, this tryptic fragment was acetylated according to the mass increase. MALDI-TOF mass spectrometry was thus demonstrated to be a fast and precise method for studying the reductive acetylation of the recombinant 1-lip E2 subunit by E1 and pyruvate. (C) 2002 Elsevier Science (USA). All rights reserved.