Figure 2

A, B and C: Representative MS spectra of N-terminal peptides from partially alpha-N-acetylated proteins. The MS-spectrum from the N-terminal peptide (doubly charged precursor) of the DnaJ homolog subfamily C member 2 (¹MLLLPSAADGR¹¹, 44% acetylated) reveals two distinguishable isotopic envelopes of the two isotopomers (i.e. the acetylated (Ac) and trideutero-acetylated forms (AcD₃)) (A). Panels B & C show MS-spectra of the N-terminal peptide (triply charged precursor) of the signal recognition particle 68 kDa protein (²AAEKQVPGGGGGGGSGGGGGSGGGGSGGGR³¹) when trideutero-acetylated (B) or ¹³C₂D₃-acetylated (C) (53% acetylated). Panel B illustrates the need for increased mass spacing by heavier isotopomers. D: Comparison of protein N-trideuteroacetylation and N-propionylation. A protein that is partially in vivo α-N-acetylated can be modified in vitro by N-trideutero-acetylation or N-propionylation. When trideutero-acetylation is used, the RP-HPLC elution profiles of the α-N-acetylated and α-N-trideutero-acetylated variants are indistinguishable and the peptide variants only segregate upon MS analysis by their 3 Da mass difference. In contrast, when N-propionylation is used the α-N-acetylated and α-N-propionylated variants segregate upon RP-HPLC, with the propionylated variant eluting at a later time because of increased hydrophobicity. E.: The use of methionine oxidation to segregate methionine-containing from non-methionine-containing N-terminal peptides. Methionine oxidation, when applied in between the primary and secondary RP-HPLC separations and after TNBS modification, causes methionine-sulfoxide containing N-terminal peptides to shift to earlier elution times on RP-HPLC. As a result the N-terminal peptides of NatB (Met-Asn, Met-Asp and Met-Glu) and NatC (Met-Ile, Met-Leu and Met-Phe) substrates, as well as of substrates of yet unidentified NATs (e.g. Met-Lys) migrate out of the primary collection intervals and are in this way enriched from the non-methionine-containing NatA substrates.