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1.
Fig. 7.

Fig. 7. From: Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo.

Deletion of yNatA (yNatAΔ) results in the specific loss of Nt-acetylation of NatA-type yeast substrate N-termini. MS-spectra from the MN- starting N-terminal peptide (doubly charged precursor) of the Ankyrin repeat-containing protein YGL242C (1MNTEGASLSEQLLDAAR17)(upper panels) and of the FK506-binding protein 1 (2SEVIEGNVKIDR13)(lower panels) demonstrate that only the latter represents a genuine yNatA substrate as it was found to be fully (Left) and 0% Nt-acetylated in the control and yNatAΔ yeast proteomes respectively, whereas the NatB-type substrate remained unaffected in its Nt-acetylation status (i.e. fully Nt-acetylated in both yeast setups).

Håvard Foyn, et al. Mol Cell Proteomics. 2013 January;12(1):42-54.
2.
Fig. 2.

Fig. 2. From: Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo.

NAT- and NPT-activity of recombinant hNaa50p toward synthetic N-terminal peptides. A, Product formation per minute per hNaa50p molecule. Purified GST-hNaa50p (75 nm with Ac-CoA and 200 nm with Prop-CoA) was incubated with oligopeptide substrates and saturated levels of Ac-CoA or Prop-CoA in acylation buffer at 37 °C for 30 min. Product formation was determined using RP-HPLC. Experiments were performed in triplicates. B, Ac-CoA- and Prop-CoA saturation curves in the presence of MLGPE oligopeptide (300 μm) and purified GST-hNaa50p (75 nm with Ac-CoA and 250 nm with Prop-CoA) in acetylation buffer at 37 °C for 30 min. Data were fitted to the Michaelis-Menten equation by Grafit 7 to determine kinetic parameters. Results shown are representative of three independent experiments.

Håvard Foyn, et al. Mol Cell Proteomics. 2013 January;12(1):42-54.
3.
Fig. 1.

Fig. 1. From: Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo.

NAT- and NPT-activity of recombinant hNaa10p toward synthetic N-terminal peptides. A, Product formation per minute per hNaa10p molecule. Purified MBP-hNaa10p (5–300 nm) was incubated with oligopeptide substrates and saturated levels of Ac-CoA or Prop-CoA in acylation buffer at 37 °C for 10 min. Product formation was determined using RP-HPLC. Experiments were performed in triplicates. B, Ac-CoA- and Prop-CoA saturation curves in the presence of EEEIA oligopeptide (300 μm) and purified MBP-hNaa10p (5 nm with Ac-CoA and 50 nm with Prop-CoA) in acetylation buffer at 37 °C for 30 min. Data were fitted to the Michaelis-Menten equation by Grafit 7 to determine kinetic parameters. Results shown are representative of three independent experiments.

Håvard Foyn, et al. Mol Cell Proteomics. 2013 January;12(1):42-54.
4.
Fig. 6.

Fig. 6. From: Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo.

NatA acts as an in vivo N-terminal propionyltransferase. Illustrative MS-spectra of the Nt-acetylated and Nt-propionylated variants of the database annotated N-terminus of the FK506-binding protein 1, a previously identified NatA substrate (1). These peptides were identified as the Nt-acetylated (upper panels) and Nt-propionylated variants (lower panels) of S2EVIEGNVKIDR13. The ion intensities of the two different N-terminal peptide forms indicate that the Nt-acetylated variant is present in nearly equal ratios (i.e. a ratio of 1.24 and 1.03 in the yNatA versus yNatAΔ setup and yNatA versus hNatA setups respectively). The Nt-propionylated variant however was exclusively identified in its 16O labeled form in the yNatA versus yNatAΔ setup while identified with a ratio of 1.92 in the yNatA versus hNatA setup, indicative for the fact that the in vivo Nt-propionylation of this protein is mediated by yNatA and that ectopically expressed hNatA complex can also act as an Nt-propionyltransferase (NPT) thereby (partially) restoring the degree of Nt-propionylation.

Håvard Foyn, et al. Mol Cell Proteomics. 2013 January;12(1):42-54.
5.
Fig. 5.

Fig. 5. From: Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo.

Propionylation as an in vivo N-terminal modification in yeast. A, Overlay of the two highest scoring MS/MS spectra of the Nt-acetylated (black) and Nt-propionylated (orange) database annotated N-terminus of the FK506-binding protein 1 (S2EVIEGNVKIDR13, Swiss-Prot accession: P20081). The shared y-ions and unique b-ions are indicated in italic and normal type interface respectively. Of note is the observed shift of 14 Da in the b-ion series because of the presence of the N-terminal modification being respectively N-terminal acetylation or N-terminal propionylation. B, Combined CID/HCD MS/MS spectrum of the Nt-propionylated database annotated N-terminus of the FK506-binding protein 1. The CID spectrum was used for identification of the peptide (S2EVIEGNVKIDR13) and is acquired during the acquisition of the HCD spectrum of the same precursor. The newly observed reporter ions indicative of N-terminal propionyation are indicated in bolt type interface. The HCD spectrum was acquired in the Orbitrap with an effective FWHM resolution greater than 7500 around m/z 400.

Håvard Foyn, et al. Mol Cell Proteomics. 2013 January;12(1):42-54.
6.
Fig. 3.

Fig. 3. From: Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo.

NAT-, NPT- and NBT-activity of immunprecipitated hNatA toward synthetic N-terminal peptides. A, Product formation per minute per hNatA complex. Human A431 cells were harvested, lysed and subjected to immunoprecipitation, using anti-hNaa15p or control antibodies. Beads containing hNatA complexes were incubated with the indicated oligopeptide substrate (200 μm) and Ac-, Prop- or But-CoA (200 μm) in acylation buffer for 60 min at 37 °C. Experiments were performed in triplicates. B, Ac-CoA-, Prop-CoA and But-CoA saturation curves in the presence of SESSS oligopeptide (200 μm) and beads containing hNatA complexes in acetylation buffer at 37 °C for 30 min. Data were fitted to the Michaelis-Menten equation by Grafit 7 to determine kinetic parameters. Results shown are representative of three independent experiments.

Håvard Foyn, et al. Mol Cell Proteomics. 2013 January;12(1):42-54.
7.
Fig. 4.

Fig. 4. From: Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo.

Outline of the differential N-terminal COFRADIC strategy used to identify NatA mediated in vivo Nt-propionylation. Proteomes from yeast cells were prepared for differential N-terminal peptide analysis. After protein S-alkylation, reduction with tris(2-carboxyethyl)phosphine (TCEP), protein S-alkylation and in vitro acetylation of primary amines, the proteomes are digested with trypsin. Pyroglutamate residues are enzymatically removed by the combined action of glutamyl cyclase and pyrolidone-carboxylic acid peptidase. Subsequently, a differential post-metabolic strategy was applied using trypsin-catalyzed C-terminal oxygen exchange. As such, the proteome digest of one sample was 16O-tagged (yNatA) and the other sample was 18O-tagged (yNatAΔ or hNatA). Equal amounts of these differentially labeled samples were mixed followed by SCX enrichment and N-terminal peptide sorting by means of N-terminal COFRADIC. Sorted fractions were subsequently analyzed by LC-MS/MS analysis. The left and right MS-spectra are representative of spectra belonging to an Nt-acetylated and Nt-propionylated N-terminus of a NatA substrate N-terminus. The Nt-acetylated N-terminus was recovered as an isotopic couple separated by 4 Da whereas the Nt-propionylated counterpart was uniquely identified in the yNatA setup (Single 16O). ‘Ac’ denotes in vivo (black) or in vitro (orange) Nt-acetylation whereas ‘Prop-’ indicates in vivo Nt-propionylation.

Håvard Foyn, et al. Mol Cell Proteomics. 2013 January;12(1):42-54.

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