Fig. 3. Functional profiling of the pyruvoyl cofactor of AMD1 by hydrazine probes.

a, Shown are three forms of AMD1 – inactive proenzyme (38 kDa) (1), catalytically competent N-terminal pyruvoyl-containing enzyme (30 kDa) expected to be probe-reactive (2), and inactivated enzyme bearing an alanine form of the cofactor (3). b, Expression and probe 1-labeling profiles of N- (left) versus C-terminal (right) FLAG-tagged AMD1 in transfected HEK293T cells. Forms (1)–(3) are indicated. c, Extracted parent ion chromatograms (left) and corresponding isotopic envelope (right) for the N-terminal pyruvoyl peptide of AMD1 labeled by probe 1. d, MS2 spectra of the N-terminal pyruvoyl peptide of AMD1 labeled by probe 1. The b- and y-ions assign the labeled site (*) to the N-terminal Ser₆₉ residue. e, Expression and probe 1-labeling profiles of endogenous AMD1 in HEK293T cells following treatment with difluoromethylornithine (DFMO). f, Expression and probe 1-labeling profiles of AMD1-transfected cells cultured in media containing 10 versus 200 µM l-methionine (left) and corresponding band intensities for each (right). Results represent average values from three biological replicates, and standard deviations are represented by error bars for the low methionine condition, where band intensities were normalized to the high methionine condition. g, From cells generated as in f, ratios for AMD1 peptides – probe 1-labeled pyruvoyl (A) and alanine (B) forms of N-terminal peptide, as well as an internal peptide (C) – in experiments comparing cells grown in low (10 µM) versus high (200 µM) methionine and compared by modifying corresponding peptides with light and heavy formaldehyde, respectively. Results are from a single experiment representative of two independent biological replicates. h, Summary table of data in g. Results shown are from a single experiment representative of two independently performed experiments.