Figure 5.
N-homocysteinylation of K294/296 within β-arrestin1/2 by Hcy and HTL. (A) Schematic workflow for the alkynyl thioester probe (AT-3)–based identification of N-homocysteinylated proteins in washed platelets treated with Hcy (100 μM) and HTL (30 μM). (B) Both β-arrestin1 and β-arrestin2 were N-homocysteinylated by Hcy and HTL in washed platelets from humans. (C) Both β-arrestin1 and β-arrestin2 were N-homocysteinylated by Hcy and HTL in washed mouse platelets. (D) MS-based identification revealed that K294 within β-arrestin1 (bovine) was the predominant target of N-homocysteinylation. Purified β-arrestin1 was incubated with HTL (10 μM), and the samples were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, in-gel trypsin digestion and LC-MS/MS analysis. N-homocysteinylated LYS residues were searched with up to 1 differential modification for homocysteine iodoacetamide-alkyne (Hcy-IAA) labeling (+302.14 Da). K294 of β-arrestin1 was accurately assigned to the expected modification on the basis of the MS/MS spectra in which the expected m/z difference between y13++ and y14++ in β-arrestin1 was observed. The structure of β-arrestin1 is shown in green, and K294 is shown in purple. (E) MS-based identification revealed that K296 within β-arrestin2 (rat) was the predominant target of N-homocysteinylation. Purified β-arrestin2 was incubated with HTL (10 μM), and the samples were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, in-gel trypsin digestion, and LC-MS/MS analysis. N-homocysteinylated LYS residues were searched with up to 1 differential modification for Hcy-IAA labeling (+302.14 Da). K296 of β-arrestin2 was accurately assigned to the expected modification on the basis of the MS/MS spectra in which the expected m/z difference between b8+ and b9+ in β-arrestin2 was observed. The structure of β-arrestin2 is shown in green, and K296 is shown in purple. (F) MEGA sequence alignment indicated that the sequence LKHEDT was conserved in β-arrestin1 (bovine) and β-arrestin2 (rat) and that K294 within β-arrestin1 (bovine) corresponded to K296 within β-arrestin2 (rat). β-arr1, β-arrestin1; β-arr2, β-arrestin2.

N-homocysteinylation of K294/296 within β-arrestin1/2 by Hcy and HTL. (A) Schematic workflow for the alkynyl thioester probe (AT-3)–based identification of N-homocysteinylated proteins in washed platelets treated with Hcy (100 μM) and HTL (30 μM). (B) Both β-arrestin1 and β-arrestin2 were N-homocysteinylated by Hcy and HTL in washed platelets from humans. (C) Both β-arrestin1 and β-arrestin2 were N-homocysteinylated by Hcy and HTL in washed mouse platelets. (D) MS-based identification revealed that K294 within β-arrestin1 (bovine) was the predominant target of N-homocysteinylation. Purified β-arrestin1 was incubated with HTL (10 μM), and the samples were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, in-gel trypsin digestion and LC-MS/MS analysis. N-homocysteinylated LYS residues were searched with up to 1 differential modification for homocysteine iodoacetamide-alkyne (Hcy-IAA) labeling (+302.14 Da). K294 of β-arrestin1 was accurately assigned to the expected modification on the basis of the MS/MS spectra in which the expected m/z difference between y13++ and y14++ in β-arrestin1 was observed. The structure of β-arrestin1 is shown in green, and K294 is shown in purple. (E) MS-based identification revealed that K296 within β-arrestin2 (rat) was the predominant target of N-homocysteinylation. Purified β-arrestin2 was incubated with HTL (10 μM), and the samples were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, in-gel trypsin digestion, and LC-MS/MS analysis. N-homocysteinylated LYS residues were searched with up to 1 differential modification for Hcy-IAA labeling (+302.14 Da). K296 of β-arrestin2 was accurately assigned to the expected modification on the basis of the MS/MS spectra in which the expected m/z difference between b8+ and b9+ in β-arrestin2 was observed. The structure of β-arrestin2 is shown in green, and K296 is shown in purple. (F) MEGA sequence alignment indicated that the sequence LKHEDT was conserved in β-arrestin1 (bovine) and β-arrestin2 (rat) and that K294 within β-arrestin1 (bovine) corresponded to K296 within β-arrestin2 (rat). β-arr1, β-arrestin1; β-arr2, β-arrestin2.

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