The enzymatic reaction for lysine acetylation and the hypothesized mechanism for lysine succinylation are indicated.

(a) MS/MS spectra of a doubly charged tryptic peptide (FTEGAFKDWGYQlAR) from isocitrate dehydrogenase (top), the synthetic succinyllysine peptide corresponding to the in vivo peptide sequence (middle) and the synthetic methylmalonyllysine peptide bearing the same peptide sequence (bottom). Insets show the precursor ion masses. The neutral loss of the Co₂ group is shown in red. (b) Extracted ion chromatograms (XICs) of the in vivo-derived isocitrate dehydrogenase peptide (top), the synthetic succinyllysine peptide bearing the same peptide sequence (middle) and a mixture of the peptide from in vivo-derived isocitrate dehydrogenase tryptic peptide and its synthetic counterpart (bottom). (c) The HPlC coelution profile of FTEGAF^SuccKDWGYQlAR (◆) and FTEGAF^MeMalKDWGYQlAR (▼), showing the different retention times of these two peptides.

(a) Specificity of anti-^SuccK antibody using dot-spot assays. Peptide libraries bearing a fixed unmodified lysine, acetyllysine or succinyllysine were spotted on nitrocellulose membrane with 10-fold dilutions. The 13-residue randomized peptide libraries have a fixed lysine residue at the seventh position: lane 1, unmodified lysine; lane 2, ^AcK; lane 3, ^SuccK. (b) Western blot analysis of recombinant E. coli isocitrate dehydrogenase (Icda), GADPH (GapA) and serine hydroxymethyltransferase (GlyA) competed with a lysine-succinylated (S) or an unmodified (U) peptide library. The same amounts of sample were loaded in both lanes for each protein. (c) Western blot analysis of lysine succinylation competed with a lysine succinylated (right) or an unmodified (left) peptide library in protein whole-cell lysates of E. coli (K-12 MG1655), S. cerevisiae (strain BY4741), D. melanogaster (S2 cells), M. musculus (C3H10 (T1/2) cells) and H. sapiens (Hela cells).

(a) MS/MS spectra of a doubly charged tryptic peptide (GGSEElYKK) from serine hydromethyltransferase (top), the synthetic succinyllysine peptide corresponding to the in vivo peptide sequence (middle) and the synthetic methylmalonyllysine peptide bearing the same peptide sequence (bottom). Insets show the precursor ion masses. The neutral loss of Co₂ group was shown in red. (b) XICs of the in vivo-derived serine hydroxymethyltransferase peptide (top), the synthetic succinyllysine peptide bearing the same peptide sequence (middle) and a mixture of the peptide from in vivo-derived serine hydroxymethyltransferase tryptic peptide and its synthetic counterpart (bottom). (c) The HPlC coelution profile of GGSEElY^SuccKK (◆) and GGSEElY^MeMalKK (▼), showing the different retention times of these two peptides.

(a) Stimulation of lysine succinylation in response to succinate. Western blot analysis (left) of whole-cell lysates from untreated (control), 80 mM and 160 mM sodium succinate treated (for 4 h) E. coli cells. Coomassie blue gel shows equal loading amounts (right). (b) MS/MS spectral numbers of H₄- and D₄-labeled succinyllysine peptides identified from E. coli that were treated with 160 mM 2,2,3,3-D₄-succinate. Results were based on Mascot sequence alignment using ion score cutoff 30 and manually verified.

(a) ClustalW (2.0.12) alignment of isocitrate dehydrogenase homologs from H. sapiens (GenInfo Identifier (GI): 5031777), M. musculus (GI: 148693874), D. melanogaster (GI: 24643268), C. elegans (GI: 71986051), S. cerevisiae (GI: 6324709) and E. coli (GI: 170080787). Conserved sites are shaded with gray and black. Conserved and nonconserved succinyllysine residues are indicated by red and blue triangles, respectively. The positions are labeled corresponding to the E. coli sequence. (b) localization of the succinyllysines and functional sites in isocitrate dehydrogenase. The three-dimensional structure was obtained from the Molecular Modeling Database (MMDB) (MMDB ID 49631) and viewed by Cn3D (v4.1). Succinylated and known functionally important sites (Uniprot ID P08200) are indicated by red and yellow arrows, respectively. The superscripts SB, NB, MB, CAT and SUCC on the labeled residues refer to: substrate binding, NADP binding, metal binding, catalytic sites and succinylation, respectively. (c) Purity of the wild-type and mutated proteins shown by SDS-PAGE gel. (d) Enzymatic activities of wild-type (control), K242R, K242E (top), K100E and K100R (bottom) isocitrate dehydrogenase mutants.