N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 99

analyzed by mass spectroscopy. Using this approach, over a dozen of N-
homocysteinylation sites have been identified in in vitro-prepared human N-Hcy
hemoglobin [214].

5.5.6 Liquid Chromatography/Mass Spectrometry Analysis of
the Site-Specific Protein

N -Homocysteinylation In Vivo

Site-specific N-homocysteinylation is assayed by using a liquid chromatography/
mass spectrometry (LC-MS) method. So far, site-specific N-homocysteinylation
in vivo has been analyzed for human serum albumin [212, 213] and fibrinogen
[215]. The sites of N-homocysteinylation identified in these proteins in vivo corre-
spond to lysine residues that are the most reactive with Hcy-thiolactone in vitro.

5.5.6.1 N -Hcy-Albumin

The LC-MS method was first used in 2004 to demonstrate that Lys525 in human
serum albumin is a predominant site of the modification by Hcy-thiolactone in vitro
and in vivo [96]. This has been achieved by the identification of
525 K*QTALVELVK 534 peptide carrying N-linked Hcy at lysine-525 ( 525 K*) in
albumin modified with Hcy-thiolactone in vitro (used at 1:1 molar ratio) as well
as in native albumin isolated from individuals with elevated plasma Hcy levels
(40-80 M). At that time, the analyses of site-specific N-homocysteinylation in vivo

required extensive sample workup and enrichment procedures [96], which pre-
cluded their routine use.
To overcome these limitations, a new LC-MS method for monitoring site-specific
albumin N-homocysteinylation directly in human plasma samples has been devel-
oped [212, 213]. To be able to detect site-specific N-homocysteinylation in vivo, it is
important to know which lysine residues in the protein are susceptible to the modifi-
cation with Hcy-thiolactone in vitro and which peptides containing N-Hcy-Lys
residues can be detected. For this purpose, N-Hcy-albumin containing 6 moles
Hcy/mol albumin is prepared by an overnight incubation of human serum albumin
(10 mg/mL) with 10 mM L -Hcy-thiolactone·HCl in 0.1 M sodium phosphate buffer,
pH 7.4, 0.1 mM EDTA at 22 C [78, 139]. One nanomole of N-Hcy-albumin is then
reduced with 45 mM DTT in 0.1 M ammonium bicarbonate, alkylated with 0.1 M
iodoacetate, and digested with trypsin at a trypsin:protein ratio of 1:50 37 C for 17 h.
Tryptic peptides are fractionated on a C18 microcolumn (ZipTip, Millipore) using 10,
30, 50, and 100 % acetonitrile [298] or on a C18 HPLC column. Each fraction is
applied on the Prespotted Anchorchip and analyzed on a MALDI-TOF Autoflex
instrument (Bruker Daltonics). Peptide identification is performed by MASCOT,
allowing for a mass increase of 57 Da due to carbamidomethylation and of 174 Da
due to N-homocysteinylation and carbamidomethylation. These analyses show that K4

μ