N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 101

digests were subjected to LC-MS analysis using a nanoscale liquid chromatogra-
phy system (EASY-nLC, Proxeon) coupled to a quadrupole-time-of-flight mass
spectrometer (micrOTOF-Q , Bruker Daltonics). Samples (10 μ L) are loaded on a

™

C18 pre-column (5 μ m i.d., Nano Separations) and separated on a C18 column

(100 m i.d., 150 mm, Nano Separations) using 0-54 % acetonitrile gradient in

0.1 % formic acid for 30 min. Data are analyzed using Data Analysis and Bio Tools
software (Bruker Daltonics).
Three albumin residues, Lys525, 137, and 212, are found to be N-homo-
cysteinylated in vivo in human plasma from CBS-deficient patients and unaffected
individuals, with Lys525 being the predominant in vivo-modified site (Fig. 5.15 ).
Albumin peptide containing N-Hcy-Lys-525 is present in essentially all analyzed
plasma samples (43 out of 44), including those which had the lowest tHcy concen-
tration (9.9

μ

M), whereas peptides containing N-Hcy-Lys-137 and N-Hcy-Lys-212
are present in CBS-deficient patients whose plasma tHcy concentration was ele-
vated, at least 34.9

μ

M, respectively [212].
To determine whether the amount of 525 K*QTALVELVK 534 peptide reflects
the extent of total serum protein N-homocysteinylation, the 651.3 m/z peptide is
quantified in tryptic digests of plasma samples from healthy individuals, who have
low levels of N-Hcy-protein, and from CBS-deficient patients, who have elevated
levels of N-Hcy-protein [115]. For quantification purposes, signal intensity of the
651.3 m/z peptide is normalized to signal intensity of a major albumin peptide
42 LVNEVTEFAK 51 (575.3 m/z).
For peptides containing N-Hcy-Lys525 and N-Hcy-Lys137,

11.0 μ M and 131 21 μ

intra-assay

variability is 10 %. Inter-assay variability determined from duplicate assays of

20 human plasma samples on two different days is 43 %. Other N-Hcy-peptides are
present in low abundance, have low signal/noise ratios, and cannot be reliably
quantified.
Normalized levels of the 651.3 m/z peptide (containing N-Hcy-Lys525) are
significantly higher in CBS-deficient patients (n ¼ 15), compared with healthy

individuals (n ¼ 29) (0.0399 0.0260 vs. 0.0102 0.0121, P ¼ 0.0007)

(Table 5.6 ).
These values suggest that about 1 % and 4 % albumin molecules are N-
homocysteinylated at Lys525 in healthy individuals and CBS-deficient patients,
respectively. The higher levels of N-homocysteinylation at Lys525 in albumin from
CBS-deficient patients reflect elevated total Hcy and N-Hcy-protein levels in these
patients [115] measured using chemical assays [297]. There is a significant correla-
tion between the albumin N-Hcy-Lys525 peptide and plasma tHcy (r ¼ 0.49,

p < 0.001) [213].

5.5.6.2 N -Hcy-Fibrinogen

Analysis of sites that are N-homocysteinylated in fibrinogen in vivo requires
purification of the protein from human plasma using the glycine precipitation
method [115, 361]. Purified fibrinogen is acetamidated, digested with trypsin, and