title: Identification of N-homocysteinylation sites in plasma proteins.
abstract: A protocol for the identification of N-homocysteinylation sites in plasma proteins is described. Human plasma or purified fibrinogen is subjected to trypsin digestion and analysis of N-Hcy-peptides by liquid chromatography/mass spectroscopy (LC/MS). Human fibrinogen is isolated from the plasma by the glycine precipitation method. Identification of N-Hcy-Lys-peptides in tryptic digests of in vivo-derived samples is facilitated by the use of N-Hcy-albumin and N-Hcy-fibrinogen synthesized in vitro from commercially available human proteins. This protocol allows identification of N-homocysteinylation sites at Lys4, Lys12, Lys137, and Lys525 in albumin directly in trypsin-digested human serum samples. N-Hcy-Lys562, N-Hcy-Lys344, and N-Hcy-Lys385 were identified in human fibrinogen from patients with cystathionine β-synthase deficiency. The protocol can be completed in 4 days.
authors.affiliation: Polish Academy of Sciences, Institute of Bioorganic Chemistry, Poznań, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Sikora
authors.forename: Łukasz
authors.initials: Ł
authors.lastname: Marczak
authors.forename: Jolanta
authors.initials: J
authors.lastname: Kubalska
authors.forename: Ałła
authors.initials: A
authors.lastname: Graban
authors.forename: Hieronim
authors.initials: H
authors.lastname: Jakubowski
mesh_terms: D001798:Blood Proteins / Q000032:analysis* / Q000378:metabolism; D006710:Homocysteine / Q000032:analysis* / Q000378:metabolism; D006712:Homocystinuria / Q000097:blood*; D006801:Humans; D010455:Peptides / Q000032:analysis* / Q000097:blood; D011499:Protein Processing, Post-Translational
chemical_list: D001798:Blood Proteins; D010455:Peptides; D006710:Homocysteine