N-terminal acetylation-specific antibodies: Specificity determination by mass spectrometry and utilization in in vitro acetylation assays.

While antibodies specifically recognizing many post-translational modifications have existed for a long time, antibodies towards the acetylated N-termini (Nt) of proteins are only just emerging. Here we further explored the potential of an antibody developed to selectively recognize Nt-acetylated Met1 of proteins (anti-Nt-Ac-Met). While partly confirming previous characterizations of this antibody, showing it to be most useful for proteins whose N-terminal sequence matches that of the N-terminal acetyltransferases NatC, NatE and NatF, we here show that this antibody may additionally be used for selective detection of Nt-acetylated Met-starting proteins of the NatB-type sequence category, i.e. MD, ME, MN, MQ. We here demonstrate that this includes the Parkinson's disease-involved protein α-synuclein, with its MDVF-starting N-terminus. Thus, this antibody could potentially be useful to detect α-synuclein Nt-Ac state in Parkinson's tissue. Further, we show an improved sensitivity and signal-to-noise detection by the anti-Nt-Ac-Met antibody in peptide dot blots by adding a fixation step to the nitrocellulose membrane after the deposition of peptides. Finally, we here tested a new methodological concept and found that Nt-Ac-specific detection by antibodies can be used to measure the output of in vitro Nt-Ac enzyme activity assays.