We expressed and purified recombinant full-length wild-type human SOD1 and its G93A and D101N variants in Expi293F cells where the initiating Met was removed and the N-terminus at Ala1 was acetylated, and identified N-terminal acetylation of the mammalian cell–purified G93A , D101N , and wild-type SOD1 using mass spectrometry (MS). Analysis of the b-ions inindicated +42.01037, +42.01014, and +42.01014 Da mass shifts representing the addition of an acetyl group to Ala1, demonstrating N-terminal acetylation in the mammalian cell–purified G93A, D101N, and wild-type SOD1.