Analysis of site-specific N-homocysteinylation of human serum albumin in vitro and in vivo using MALDI-ToF and LC-MS/MS mass spectrometry

Design of protein homocystamides with enhanced tumor uptake properties for 19F magnetic resonance imaging

“…Under N-homocysteinylation of HSA by HTL in vivo, only three of the 59 protein lysine residues appeared to be modified (Lys-525, Lys-137 and Lys-212). 49 Accordingly, one approach to site-specific protein fluorination may be through chemical modification of the -amino group of specific lysine residues using fluorinated HTL derivatives. This paper describes the development of new fluorinated homocysteine derivatives as potential tags for 19 F MRI.…”

“…49 Changes in molecular mass of albumin modified with PFT-HTL was monitored with MALDI-ToF mass spectrometry. Molecular mass of commercially available serum albumin A3782 assigned in our MALDI-ToF experiment was 66.47 kDa, deconvoluted molecular mass of modified albumin using the same system was 67.47 kDa.…”

“…48 N-Homocysteinylation of HSA by homocysteine thiolactone (HTL) in vivo involves modification of only three of the 59 protein lysine residues (Lys-525, Lys-137 and Lys-212). 49 On the other hand, eight different lysine modifications sites (Lys-525, Lys-212, Lys-205, Lys-159, Lys-137, Lys-12 and Lys-4) were identified in vitro, suggesting that the preferred site of modification is dependent on the HTL concentration. Therefore, it is possible to find optimal conditions for site-specific introduction of fluorine labels into the protein via its N-homocysteinylation by fluorinated HTL derivatives.…”

Homocysteine in Protein Structure/Function and Human Disease

“…For example, at pH 7.4 and 3.2 Physicochemical Properties temperature 37 C, Hcy-thiolactone modifies proteins by forming N-Hcy-protein adducts, in which Hcy is N-linked to the ε-amino group of protein lysine residues as shown in Reaction 3.4 [68,73,78]. Indeed, N-linked Hcy is found in albumin [78,96,212,213], hemoglobin [214], fibrinogen [116,215], and cytochrome c [136] only on lysine residues. Indeed, N-linked Hcy is found in albumin [78,96,212,213], hemoglobin [214], fibrinogen [116,215], and cytochrome c [136] only on lysine residues.…”

“…Mass spectrometric analyses have also identified that only ε-amino groups of internal lysine residues, but not α-amino group of the N-terminal amino acid, are targets for Hcy-thiolactone modification in human serum albumin [96,212,213], hemoglobin [68,214], cytochrome c [298], fibrinogen [175,215], and dynein [299].…”