Comparison of Protein N-Homocysteinylation in Rat Plasma under Elevated Homocysteine Using a Specific Chemical Labeling Method

Abstract

Elevated blood concentrations of homocysteine have been well established as a risk factor for cardiovascular diseases and neuropsychiatric diseases, yet the etiologic relationship of homocysteine to these disorders remains poorly understood. Protein N-homocysteinylation has been hypothesized as a contributing factor; however, it has not been examined globally owing to the lack of suitable detection methods. We recently developed a selective chemical method to label N-homocysteinylated proteins with a biotin-aldehyde tag followed by Western blotting analysis, which was further optimized in this study. We then investigated the variation of protein N-homocysteinylation in plasma from rats on a vitamin B12 deficient diet. Elevated "total homocysteine" concentrations were determined in rats with a vitamin B12 deficient diet. Correspondingly, overall levels of plasma protein N-homocysteinylation displayed an increased trend, and furthermore, more pronounced and statistically significant changes (e.g., 1.8-fold, p-value: 0.03) were observed for some individual protein bands. Our results suggest that, as expected, a general metabolic correlation exists between "total homocysteine" and N-homocysteinylation, although other factors are involved in homocysteine/homocysteine thiolactone metabolism, such as the transsulfuration of homocysteine by cystathionine β-synthase or the hydrolysis of homocysteine thiolactone by paraoxonase 1 (PON1), may play more significant or direct roles in determining the level of N-homocysteinylation.

Full text

molecules
Article
Comparison of Protein N-Homocysteinylation in Rat
Plasma under Elevated Homocysteine Using
a Specific Chemical Labeling Method
Tianzhu Zang 1, Ligi Paul Pottenplackel 2, Diane E. Handy 3, Joseph Loscalzo 3, Shujia Dai 1,
Richard C. Deth 4, Zhaohui Sunny Zhou 1, * and Jisheng Ma 1, 5, *
1Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology,
Northeastern University, Boston, MA 02115, USA; zangt@mail.med.upenn.edu (T.Z.);
daishujia@gmail.com (S.D.)
2Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111, USA;
Ligi.Paul_Pottenplackel@tufts.edu
3
Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA;
dhandy@rics.bwh.harvard.edu (D.E.H.); jloscalzo@partners.org (J.L.)
4Department of Pharmaceutical Sciences, Nova Southeastern University, Fort Lauderdale, FL 33314, USA;
rdeth@nova.edu
5School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325035, China
*Correspondence: z.zhou@northeastern.edu (Z.S.Z.); jishengma@163.com (J.M.);
Tel.: +1-617-373-4818 (Z.S.Z.); +86-130-3921-6705 (J.M.)
Academic Editor: Derek J. McPhee
Received: 16 August 2016; Accepted: 5 September 2016; Published: 8 September 2016
Abstract:
Elevated blood concentrations of homocysteine have been well established as a risk
factor for cardiovascular diseases and neuropsychiatric diseases, yet the etiologic relationship
of homocysteine to these disorders remains poorly understood. Protein N-homocysteinylation
has been hypothesized as a contributing factor; however, it has not been examined globally
owing to the lack of suitable detection methods. We recently developed a selective chemical
method to label N-homocysteinylated proteins with a biotin-aldehyde tag followed by Western
blotting analysis, which was further optimized in this study. We then investigated the variation
of protein N-homocysteinylation in plasma from rats on a vitamin B
12
deficient diet. Elevated
“total homocysteine” concentrations were determined in rats with a vitamin B
12
deficient diet.
Correspondingly, overall levels of plasma protein N-homocysteinylation displayed an increased
trend, and furthermore, more pronounced and statistically significant changes (e.g., 1.8-fold, p-value:
0.03) were observed for some individual protein bands. Our results suggest that, as expected,
a general metabolic correlation exists between “total homocysteine” and N-homocysteinylation,
although other factors are involved in homocysteine/homocysteine thiolactone metabolism, such as
the transsulfuration of homocysteine by cystathionine
β
-synthase or the hydrolysis of homocysteine
thiolactone by paraoxonase 1 (PON1), may play more significant or direct roles in determining the
level of N-homocysteinylation.
Keywords:
hyperhomocysteinemia; cardiovascular disease; neuropsychiatric disease; protein
N-homocysteinylation; plasma; biotin-aldehyde; Western blotting
1. Introduction
An abnormally increased concentration of homocysteine (Hcy) in blood or urine, i.e.,
hyperhomocysteinemia or homocystinuria, has been well recognized as a risk factor for cardiovascular,
neuropsychiatric diseases, and other conditions [
1
–
6
]. As illustrated in Figure 1, in mammals, Hcy can
Molecules 2016,21, 1195; doi:10.3390/molecules21091195 www.mdpi.com/journal/molecules

Molecules 2016,21, 1195 2 of 12
be metabolized through two main pathways: methylation and transsulfuration [
7
–
11
]. In addition,
Hcy can also be converted back to S-adenosyl-homocysteine (AdoHcy or SAH) because of the
reversible transformation catalyzed by AdoHcy hydrolase (EC 3.3.1.1) [
12
,
13
]. It has been reported
that methionine-rich diets (e.g., animal proteins), genetic defects of enzymes such as cystathionine
β
-synthase (CBS), deficiencies of nutritional factors (folate, vitamin B
6
and B
12
), or a combination of
such factors can lead to an increase of plasma “total homocysteine” (“total Hcy”) [
7
,
8
,
14
–
19
], which
includes protein S-homocysteinylation (disulfides) and some free small molecule forms (e.g., free
Hcy and its mixed disulfides with cysteine or glutathione) [
3
,
20
]. However, the pathophysiological
consequences of homocysteinemia remain unclear.
Molecules 2016, 21, 1195 2 of 12
reversible transformation catalyzed by AdoHcy hydrolase (EC 3.3.1.1) [12,13]. It has been reported
that methionine-rich diets (e.g., animal proteins), genetic defects of enzymes such as cystathionine
β-synthase (CBS), deficiencies of nutritional factors (folate, vitamin B6 and B12), or a combination of
such factors can lead to an increase of plasma “total homocysteine” (“total Hcy”) [7,8,14–19], which
includes protein S-homocysteinylation (disulfides) and some free small molecule forms (e.g., free
Hcy and its mixed disulfides with cysteine or glutathione) [3,20]. However, the pathophysiological
consequences of homocysteinemia remain unclear.
Figure 1. Metabolism of homocysteine in mammals. AdoHcy, S-adenosylhomocysteine; AdoMet,
S-adenosylmethionine; ATP, adenosine-5′-triphosphate; CBS, cystathionine β-synthase; Hcy, homocysteine;
Hcy TL, homocysteine thiolactone; MetRS, methionyl-tRNA synthetase; MS, methionine synthase;
PON1, paraoxonase 1; SAHH, S-adenosylhomocysteine hydrolase; THF, tetrahydrofolate.
Possible mechanisms of Hcy toxicity have been proposed, such as the induction of oxidative
stress and the ensuing-alteration of protein structure and loss of protein function, or the inhibition of
transmethylation by the accumulation of AdoHcy—the common methylation product and a potent
inhibitor for most methyltransferases [21–26]. Another possibility is that homocysteine is covalently
attached to proteins, including S-homocysteinylation by forming mixed disulfides with cysteine residues
and N-homocysteinylation by forming homocystamides (Figure 2) [27–29]. Protein S-homocysteinylation
has been identified in several plasma proteins, for example, S-homocysteinylated transthyretin
[30,31]. It is worth noting that “total Hcy” is a misnomer (historical usage), as it includes more than
70% of protein S-homocysteinylation (disulfides), but excludes other important Hcy species, such as
protein N-homocysteinylation in human plasma [3,20,22]. First reported by Jakubowski, protein
N-homocysteinylation results from the non-enzymatic acylation of the amino groups in proteins
(either on the side chain of lysines and/or the N-termini) by homocysteine thiolactone (Hcy thiolactone
or Hcy TL), which is produced as a byproduct from the editing process of methionyl-tRNA synthetase
(Figures 1 and 2) [29,32–34].
Figure 1.
Metabolism of homocysteine in mammals. AdoHcy, S-adenosylhomocysteine; AdoMet,
S-adenosylmethionine; ATP, adenosine-5
0
-triphosphate; CBS, cystathionine
β
-synthase; Hcy, homocysteine;
Hcy TL, homocysteine thiolactone; MetRS, methionyl-tRNA synthetase; MS, methionine synthase;
PON1, paraoxonase 1; SAHH, S-adenosylhomocysteine hydrolase; THF, tetrahydrofolate.
Possible mechanisms of Hcy toxicity have been proposed, such as the induction of oxidative
stress and the ensuing-alteration of protein structure and loss of protein function, or the inhibition
of transmethylation by the accumulation of AdoHcy—the common methylation product and a
potent inhibitor for most methyltransferases [
21
–
26
]. Another possibility is that homocysteine is
covalently attached to proteins, including S-homocysteinylation by forming mixed disulfides with
cysteine residues and N-homocysteinylation by forming homocystamides (Figure 2) [
27
–
29
]. Protein
S-homocysteinylation has been identified in several plasma proteins, for example, S-homocysteinylated
transthyretin [
30
,
31
]. It is worth noting that “total Hcy” is a misnomer (historical usage), as it includes
more than 70% of protein S-homocysteinylation (disulfides), but excludes other important Hcy species,
such as protein N-homocysteinylation in human plasma [
3
,
20
,
22
]. First reported by Jakubowski,
protein N-homocysteinylation results from the non-enzymatic acylation of the amino groups in proteins
(either on the side chain of lysines and/or the N-termini) by homocysteine thiolactone (Hcy thiolactone
or Hcy TL), which is produced as a byproduct from the editing process of methionyl-tRNA synthetase
(Figures 1and 2) [29,32–34].

Molecules 2016,21, 1195 3 of 12
Molecules 2016, 21, 1195 3 of 12
Figure 2. Formation of protein homocysteinylation and detection of N-homocysteinylation via
selective tagging with aldehydes. (A) Reversible formation of S-homocysteinylation (disulfide); and
(B) irreversible formation of N-homocysteinylation (amide) and its detection.
In comparison to S-homocysteinylation, protein N-homocysteinylation is irreversible (chemically
stable with no enzyme found to reverse the process) and, thus, may accumulate in proteins, particularly
in long-lived ones [35,36]. In human plasma, N-homocysteinylation was reported ~0.2–0.5 μM,
and the ratio of “total Hcy” to total N-homocysteinylation was found to be 7:1 to 10:1 [27,37,38].
Several human proteins have been examined more closely; human serum albumin and hemoglobin
contain 0.3% and 0.6% N-homocysteinylation (mol of modification/mol of total protein), and 0.06%
and 1.0% S-homocysteinylation, respectively [39]. Another publication reported that the levels of
N-homocysteinylated (N-Hcy) proteins in plasma were increased due to the mutations or deletions
in the cystathionine β-synthase (CBS) or methylenetetrahydrofolate reductase genes in mice [40].
Like other protein modifications, protein N-homocysteinylation may affect protein cleavage,
crosslinking, aggregation, autoimmune response, and function [41–52]. In addition, thiols are highly
reactive and may undergo myriad transformations under physiological conditions (such as redox,
alkylation, and even desulfurization) [53–57]. One example is homocystamide-induced protein oxidative
damage by triggering the formation of free radicals, discovered by Strongin’s laboratory [58]. It has
also been observed that the formation of protein aggregates from N-homocysteinylated acidic proteins
(e.g., α-lactalbumin) induces the tertiary structural changes and functional alterations [59]. Recently,
it was reported that N-Hcy proteins affected gene expression in human vascular endothelial cells,
which is related to cardiovascular development and neurological disease [60]. Thus, Jakubowski and
others have hypothesized that protein N-homocysteinylation is an important contributor to the
pathological consequences of hyperhomocysteinemia, either together with or independent of elevated
concentrations of plasma “total Hcy” (free Hcy and disulfides) [29,39,45,61].
Methods for the quantification of N-Hcy proteins have been developed. The first and most
commonly used is the complete chemical hydrolysis of proteins, followed by subsequent analysis of
free homocysteine using HPLC coupled with fluorescence or UV detection [37,62]; however, the
modified sites cannot be identified based on current methods of amino acid analysis. Alternatively,
an immunological assay (ELISA and dot blotting) using polyclonal antibodies was reported [36,63],
but the specificity remains to be established.
Figure 2.
Formation of protein homocysteinylation and detection of N-homocysteinylation via
selective tagging with aldehydes. (
A
) Reversible formation of S-homocysteinylation (disulfide); and
(B) irreversible formation of N-homocysteinylation (amide) and its detection.
In comparison to S-homocysteinylation, protein N-homocysteinylation is irreversible (chemically
stable with no enzyme found to reverse the process) and, thus, may accumulate in proteins, particularly
in long-lived ones [
35
,
36
]. In human plasma, N-homocysteinylation was reported ~0.2–0.5
µ
M,
and the ratio of “total Hcy” to total N-homocysteinylation was found to be 7:1 to 10:1 [
27
,
37
,
38
].
Several human proteins have been examined more closely; human serum albumin and hemoglobin
contain 0.3% and 0.6% N-homocysteinylation (mol of modification/mol of total protein), and 0.06%
and 1.0% S-homocysteinylation, respectively [
39
]. Another publication reported that the levels of
N-homocysteinylated (N-Hcy) proteins in plasma were increased due to the mutations or deletions in
the cystathionine β-synthase (CBS) or methylenetetrahydrofolate reductase genes in mice [40].
Like other protein modifications, protein N-homocysteinylation may affect protein cleavage,
crosslinking, aggregation, autoimmune response, and function [
41
–
52
]. In addition, thiols are highly
reactive and may undergo myriad transformations under physiological conditions (such as redox,
alkylation, and even desulfurization) [
53
–
57
]. One example is homocystamide-induced protein
oxidative damage by triggering the formation of free radicals, discovered by Strongin’s laboratory [
58
].
It has also been observed that the formation of protein aggregates from N-homocysteinylated acidic
proteins (e.g.,
α
-lactalbumin) induces the tertiary structural changes and functional alterations [
59
].
Recently, it was reported that N-Hcy proteins affected gene expression in human vascular endothelial
cells, which is related to cardiovascular development and neurological disease [
60
]. Thus, Jakubowski
and others have hypothesized that protein N-homocysteinylation is an important contributor to the
pathological consequences of hyperhomocysteinemia, either together with or independent of elevated
concentrations of plasma “total Hcy” (free Hcy and disulfides) [29,39,45,61].
Methods for the quantification of N-Hcy proteins have been developed. The first and most
commonly used is the complete chemical hydrolysis of proteins, followed by subsequent analysis
of free homocysteine using HPLC coupled with fluorescence or UV detection [
37
,
62
]; however, the
modified sites cannot be identified based on current methods of amino acid analysis. Alternatively,
an immunological assay (ELISA and dot blotting) using polyclonal antibodies was reported [
36
,
63
],
but the specificity remains to be established.

Molecules 2016,21, 1195 4 of 12
Until now, lack of a systematic analysis of N-Hcy proteins in complex systems has prevented a more
thorough understanding of molecular and pathobiological consequences in hyperhomocysteinemia.
Toward this end, we have developed a chemical method to selectively derivatize N-Hcy groups
with different aldehyde tags under mildly acidic conditions [
64
]. For example, by introducing a
biotin-containing aldehyde tag onto the N-homocystamide group (Figure 2B), Western blotting coupled
with a chemiluminescence assay can be used to both detect and quantify N-homocysteinylation
of different proteins. In comparison to antibodies against N-Hcy proteins, the biotin-aldehyde is
commercially available and inexpensive. Hence, our method makes it feasible for global profiling and
quantitative analysis of N-Hcy proteins in complex systems [
64
], including proteomic studies from
other laboratories [
65
,
66
]. In this study, we investigated changes in protein N-homocysteinylation
associated with variation of “total Hcy” concentrations in plasma from rats. Taken together,
this proteomic study reveals that protein N-homocysteinylation may be affected by homocysteine
metabolism and, furthermore, opens new avenues by which to discover potential biomarkers and to
understand better the underlying molecular mechanisms.
2. Results and Discussions
2.1. Optimized Conditions for Aldehyde Tag Labeling
As we previously demonstrated, the coupling between aldehydes and N-homocystamide is highly
specific under mildly acidic conditions (pH 2 to 4), because competing amines are protonated and
rendered inactive [
64
,
67
]. However, the pH values of the loading buffer and running buffer for sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are around 6.8 and 8.3, respectively;
under these conditions, significant percentages of amines in proteins are present in the neutral form,
favoring the formation of a protein Schiff base with the excess aldehyde labeling reagent. Hence,
in this study, cysteamine was added to the solution to quench the excess aldehyde reagent. As a
beta-amino thiol, cysteamine reacts with aldehyde to form 1,3-thiazolidine at pH 3.0 (Figure S2 in
Supplementary Materials). Under our conditions, after quenching with cysteamine, no aldehyde
reagent was left in the solution to react with free amino groups during the subsequent sample handling
steps, thereby eliminating non-specific labeling (Figure 3). Moreover, compared to antibody-based
assays, our labeling method uses a commercially available biotin-aldehyde and streptavidin without
the need of more expensive primary and secondary antibodies, and moreover, only requires one-step
Western blotting (i.e., no need for secondary antibodies). Altogether, our method significantly reduces
both cost and time, and also improves both accuracy and reproducibility.
Molecules 2016, 21, 1195 4 of 12
Until now, lack of a systematic analysis of N-Hcy proteins in complex systems has prevented a more
thorough understanding of molecular and pathobiological consequences in hyperhomocysteinemia.
Toward this end, we have developed a chemical method to selectively derivatize N-Hcy groups
with different aldehyde tags under mildly acidic conditions [64]. For example, by introducing a
biotin-containing aldehyde tag onto the N-homocystamide group (Figure 2B), Western blotting coupled
with a chemiluminescence assay can be used to both detect and quantify N-homocysteinylation of
different proteins. In comparison to antibodies against N-Hcy proteins, the biotin-aldehyde is
commercially available and inexpensive. Hence, our method makes it feasible for global profiling
and quantitative analysis of N-Hcy proteins in complex systems [64], including proteomic studies from
other laboratories [65,66]. In this study, we investigated changes in protein N-homocysteinylation
associated with variation of “total Hcy” concentrations in plasma from rats. Taken together, this
proteomic study reveals that protein N-homocysteinylation may be affected by homocysteine
metabolism and, furthermore, opens new avenues by which to discover potential biomarkers and to
understand better the underlying molecular mechanisms.
2. Results and Discussions
2.1. Optimized Conditions for Aldehyde Tag Labeling
As we previously demonstrated, the coupling between aldehydes and N-homocystamide is
highly specific under mildly acidic conditions (pH 2 to 4), because competing amines are protonated
and rendered inactive [64,67]. However, the pH values of the loading buffer and running buffer for
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are around 6.8 and 8.3,
respectively; under these conditions, significant percentages of amines in proteins are present in the
neutral form, favoring the formation of a protein Schiff base with the excess aldehyde labeling reagent.
Hence, in this study, cysteamine was added to the solution to quench the excess aldehyde reagent.
As a beta-amino thiol, cysteamine reacts with aldehyde to form 1,3-thiazolidine at pH 3.0 (Figure S2 in
Supplementary Materials). Under our conditions, after quenching with cysteamine, no aldehyde
reagent was left in the solution to react with free amino groups during the subsequent sample handling
steps, thereby eliminating non-specific labeling (Figure 3). Moreover, compared to antibody-based
assays, our labeling method uses a commercially available biotin-aldehyde and streptavidin without
the need of more expensive primary and secondary antibodies, and moreover, only requires one-step
Western blotting (i.e., no need for secondary antibodies). Altogether, our method significantly reduces
both cost and time, and also improves both accuracy and reproducibility.
Figure 3. Fluorescence intensities of rhodamine-aldehyde labeled N-Hcy myoglobin with and without
cysteamine quenching reaction. Lane 1: 7.8 μM modified myoglobin including 3.1 μM N-Hcy myoglobin
and 4.7 μM native myoglobin with cysteamine (500 μM) quenching; Lane 2: 7.8 μM modified myoglobin
including 3.1 μM N-Hcy myoglobin and 4.7 μM native myoglobin without cysteamine quenching.
Figure 3.
Fluorescence intensities of rhodamine-aldehyde labeled N-Hcy myoglobin with and without
cysteamine quenching reaction. Lane 1: 7.8
µ
M modified myoglobin including 3.1
µ
MN-Hcy
myoglobin and 4.7
µ
M native myoglobin with cysteamine (500
µ
M) quenching; Lane 2: 7.8
µ
M
modified myoglobin including 3.1
µ
MN-Hcy myoglobin and 4.7
µ
M native myoglobin without
cysteamine quenching.

Molecules 2016,21, 1195 5 of 12
2.2. Increased Level of Protein N-Homocysteinylation in Plasma from Rats on B12 Deficient Diet
As described in Figure 1, nutrients such as folate, vitamin B
12
and B
6
affect the concentration of
homocysteine [
7
,
17
]. A diet deficient in vitamin B
12
has been observed to cause hyperhomocysteinemia
in humans and animals [
68
–
71
]. Rats on vitamin B
12
-deficient or control diets (six each) were analyzed
in this work. The plasma concentration of “total Hcy” was 31.1
±
10.0 (standard deviation, SD)
and 4.7
±
0.8
µ
M for rats on B
12
-deficient and control diets, respectively (Table 1), a difference
of 6.6-fold. As expected, the overall level of protein N-homocysteinylation shows an increased
trend with 1.3-fold rising (see overall intensity in Table 1and Figure S3), while the abundance of
plasma proteins (total and individual) remained about the same (Coomassie blue image in Figure 4).
More strikingly, several individual bands displayed much larger differences in protein modifications
(Figure 4and Table 1). For example, normalized to the control group (1.0
±
0.4), band 11 has a ratio
of 1.8
±
0.7 for B
12
-defecient rats with a p-value of 0.03 (Table 1). Moreover, it should be pointed out that
plasma samples were blinded to treatment and the nature of the samples revealed only after all analysis
had been completed as shown above. Thus, our method is well suited for large scale quantification of
protein N-homocysteinylation in biological samples. So far, this method has been applied to determine
the relative levels of N-homocysteinylation in serum from autistic children with elevated levels of Hcy
to investigate the relationship between N-homocysteinylation and neurophysiological disorders [66],
in embryos from mice with the folate-responsive neural tube defects to check the correlation between
folate and N-homocysteinylation in the embryos [
65
], and in plasma from mice with heterozygous
deficiency (Cbs
+/−
) of cystathionine
β
-synthase (CBS) to study the correlation between CBS deficiency
and protein N-homocysteinylation (preliminary data from our laboratories shown in Figure S4) [
72
,
73
].
Further studies would be focused on the identification of N-Hcy proteins and modified sites in order
to discover the potential N-Hcy protein biomarkers. This assay could be accomplished by enrichment
of N-Hcy protein digests using aldehyde resin coupled with liquid chromatography tandem mass
spectrometry (LC-MS/MS) analysis [
64
]. For future work, quantitative mass spectrometry approaches
such as isotopic labeling (e.g., iTRAQ) can be combined with ours [
74
]. Such a combination should
also allow us to perform both identification of the modified sites and quantification at the same time.
Table 1.
Chemiluminescence intensities of biotin-labeled proteins (N-homocysteinylation) and “total
homocysteine” concentration of rat plasma.
B12 Deficiency * Control Diet
Chemiluminescence Intensity
Band Mean ±S.D. Mean ±S.D. p-Value
1 1.2 ±0.3 1.0 ±0.4 0.3
2 1.3 ±0.4 1.0 ±0.3 0.2
3 1.3 ±0.4 1.0 ±0.3 0.2
4 1.2 ±0.3 1.0 ±0.2 0.2
5 1.2 ±0.2 1.0 ±0.2 0.09
6 1.3 ±0.2 1.0 ±0.2 0.03
7 1.4 ±0.3 1.0 ±0.2 0.009
8 1.4 ±0.2 1.0 ±0.3 0.04
9 1.2 ±0.3 1.0 ±0.3 0.2
10 1.5 ±0.6 1.0 ±0.4 0.09
11 1.8 ±0.7 1.0 ±0.4 0.03
Overall 1.3 ±0.3 1.0 ±0.2 0.08
“Total homocysteine” (µM)
31.1 ±10.0 4.7 ±0.8 5.00 ×10−5
*: six samples in each group were analyzed in duplicate; overall intensity is for all proteins in the complete
lanes. Intensities have been normalized to those from control diet; p-value < 0.05 is considered a statistically
significant change (highlighted in italic); S.D.: standard deviation.

Molecules 2016,21, 1195 6 of 12
Molecules 2016, 21, 1195 6 of 12
Figure 4. Gel images of plasma proteins from rats on B12-deficient and control diets. Left image:
Coomassie blue staining showing total protein loading; right image: chemiluminescence from Western
blotting showing the levels of biotin-labeling (protein N-homocysteinylation). Lanes 1, 2, and 3: plasma
from individual rats on control diet; lanes 4 and 5: plasma from individual rats on a B12-deficient
diet. Proteins were divided into eleven individual protein bands (indicated by the arrows) for
subsequent analysis.
2.3. Possible Contributing Factors to N-Homocysteinylation
As shown in our work, whereas there were increases in N-homocysteinylation when the blood
concentrations of “total Hcy” (disulfides) were elevated, the magnitude of the former is smaller. This
finding is not unexpected, considering the formation of “total Hcy” (free Hcy and disulfides) and
N-homocysteinylation are influenced by multiple and different factors. As illustrated in Figure 1,
except the nutritional factors, the activities of different enzymes which involve in the conversion of
Hcy to methionine, cysteine and Hcy thiolactone could also contribute to the variation of protein
N-homocysteinylation. Hcy thiolactone can be hydrolyzed back to Hcy by paraoxonase 1 (PON1, EC
3.1.8.1) [35]. Therefore, activity of PON1 likely plays a direct and significant role in Hcy thiolactone
metabolism and, hence, N-homocysteinylation. For example, PON1 is present in serum, and there is little
protein synthesis in serum (hence, little formation of Hcy thiolactone) [75,76]. Thus, the steady-state
concentration of Hcy thiolactone is extremely low (0.1–26 nM), while “total Hcy” is in the 5–15 μM
range [22]; as such, the level of N-homocysteinylation is expected to be lower than the “total Hcy”, as
we observed here. Conversely, in tissues and within cellular compartments, where protein synthesis is
more active, Hcy thiolactone formation is likely to be higher as well. Ultimately, the counterbalance of
the formation and hydrolysis of Hcy thiolactone, not simply the concentration of homocysteine,
determines the level of N-homocysteinylation. In addition, as mentioned in Section 1, the inactivation
of cystathionine β-synthase (CBS) in transgenic mice (Cbs−/−) caused 50- to 140-fold elevation of “total Hcy”
and six- to 10-fold increase of N-Hcy proteins in serum [40]. As such, patients with genetically-deficient
enzymes could cause a more striking increase of N-Hcy proteins, and N-Hcy proteins would be more
effectively identified using LC-MS/MS. Recently, N-Hcy fibrinogen and its modified sites have been
identified in the plasma from CBS-deficient patients [77]. Based on our results and the published
reports, we propose that differences on enzyme activity (e.g., PON1 and CBS) are likely to alter
plasma N-homocysteinylation status more distinctly, which can be tested using our method. Finally,
given the simplicity, low-cost, and robustness of our method, our method can be easily employed for
clinical applications [66].
3. Materials and Methods
3.1. Plasma
Plasma from rats on vitamin B12-deficient and control diets (six in each group) were from Dr.
Ligi Paul at Tufts University. All animal procedures were approved by the Institutional Animal Care
Figure 4.
Gel images of plasma proteins from rats on B
12
-deficient and control diets. Left image:
Coomassie blue staining showing total protein loading; right image: chemiluminescence from Western
blotting showing the levels of biotin-labeling (protein N-homocysteinylation). Lanes 1, 2, and 3: plasma
from individual rats on control diet; lanes 4 and 5: plasma from individual rats on a B
12
-deficient
diet. Proteins were divided into eleven individual protein bands (indicated by the arrows) for
subsequent analysis.
2.3. Possible Contributing Factors to N-Homocysteinylation
As shown in our work, whereas there were increases in N-homocysteinylation when the blood
concentrations of “total Hcy” (disulfides) were elevated, the magnitude of the former is smaller. This
finding is not unexpected, considering the formation of “total Hcy” (free Hcy and disulfides) and
N-homocysteinylation are influenced by multiple and different factors. As illustrated in Figure 1,
except the nutritional factors, the activities of different enzymes which involve in the conversion of
Hcy to methionine, cysteine and Hcy thiolactone could also contribute to the variation of protein
N-homocysteinylation. Hcy thiolactone can be hydrolyzed back to Hcy by paraoxonase 1 (PON1,
EC 3.1.8.1) [
35
]. Therefore, activity of PON1 likely plays a direct and significant role in Hcy thiolactone
metabolism and, hence, N-homocysteinylation. For example, PON1 is present in serum, and there
is little protein synthesis in serum (hence, little formation of Hcy thiolactone) [
75
,
76
]. Thus, the
steady-state concentration of Hcy thiolactone is extremely low (0.1–26 nM), while “total Hcy” is in
the 5–15
µ
M range [
22
]; as such, the level of N-homocysteinylation is expected to be lower than the
“total Hcy”, as we observed here. Conversely, in tissues and within cellular compartments, where
protein synthesis is more active, Hcy thiolactone formation is likely to be higher as well. Ultimately,
the counterbalance of the formation and hydrolysis of Hcy thiolactone, not simply the concentration of
homocysteine, determines the level of N-homocysteinylation. In addition, as mentioned in Section 1,
the inactivation of cystathionine
β
-synthase (CBS) in transgenic mice (Cbs
−/−
) caused 50- to 140-fold
elevation of “total Hcy” and six- to 10-fold increase of N-Hcy proteins in serum [
40
]. As such, patients
with genetically-deficient enzymes could cause a more striking increase of N-Hcy proteins, and N-Hcy
proteins would be more effectively identified using LC-MS/MS. Recently, N-Hcy fibrinogen and its
modified sites have been identified in the plasma from CBS-deficient patients [
77
]. Based on our results
and the published reports, we propose that differences on enzyme activity (e.g., PON1 and CBS) are
likely to alter plasma N-homocysteinylation status more distinctly, which can be tested using our
method. Finally, given the simplicity, low-cost, and robustness of our method, our method can be
easily employed for clinical applications [66].

Molecules 2016,21, 1195 7 of 12
3. Materials and Methods
3.1. Plasma
Plasma from rats on vitamin B
12
-deficient and control diets (six in each group) were from
Dr. Ligi Paul at Tufts University. All animal procedures were approved by the Institutional Animal
Care and Use Committee of the Jean Mayer USDA Human Nutrition Research Center on Aging at
Tufts University (Protocol No. SE-56) and conducted according to the Guide for the Care and Use of
Laboratory Animals (1996). Measurement of “total Hcy” in plasma was performed as reported [
78
].
Protein concentrations of plasma were assayed according to the Bradford method using concentrated
dye (500-0006) from Bio-Rad (Hercules, CA, USA); and bovine serum albumin (A3059) purchased from
Sigma-Aldrich (St. Louis, MO, USA) was used as a standard.
3.2. Chemicals and Reagents
Horseradish peroxidase streptavidin (SA-5004) was from Vector Laboratories (Burlingame, CA,
USA). SuperSignal West Pico chemiluminescent substrate was from Thermo Scientific (Rockford, IL,
USA). Myoglobin from equine skeletal muscle (M0630) was from Sigma-Aldrich. N-Homocysteinylated
myoglobin was prepared as we previously reported [
64
]. Biotinyl-Asp-Glu-Val-Asp-aldehyde (CAS
registry number: 178603-73-1) was from Bachem Americas (N-1470, Torrance, CA, USA; see Figure S1
for its structure in Support Information). Rhodamine-aldehyde (10 mM) in 50% 200-proof ethanol was
synthesized as previously described [
64
]. EZ-Run prestained protein ladder (BP3603) was from Fisher
Scientific. Natural unstained protein ladder (161-0317) was from Bio-Rad. Biotinylated protein markers
were from Sigma-Aldrich (B2787) and Bio-Rad (161-0319), respectively. All reagents were ACS grade
and used as received without further purification. All incubations were carried out in an Eppendorf
Thermomixer
®
(Eppendorf North America, Hauppauge, NY, USA) at 25
◦
C unless specified otherwise.
3.3. Optimization of Labeling with Cysteamine Quenching
Hcy thiolactone-modified myoglobin (13
µ
M including 5.2
µ
MN-Hcy myoglobin (~40%) and
7.8
µ
M native myoglobin) was incubated with 200
µ
M Rhodamine-aldehyde (pH 3), containing
50 mM citric acid, 500
µ
M tris(2-carboxyethyl)phosphine (TCEP), at 25
◦
C in the dark for 8 h [
64
].
Aliquots (20
µ
L) were removed and stored at 4
◦
C. Cysteamine (15 mM, 1
µ
L) was added to the
remaining solution to quench the excess aldehyde for 3–14 h. Solutions (12
µ
L) with or without
cysteamine quenching were mixed with loading buffer (8
µ
L, 5% SDS, and 25% glycerol) for SDS-PAGE
analysis. Gels were analyzed using a Molecular Dynamics Storm840 imaging system (GE Healthcare,
Piscataway, NJ, USA). Fluorescence was recorded with an excitation wavelength at 450 nm and
an emission wavelength at 520 nm using a Strom Scanner Control version 5.03 (Amersham Bioscience,
Piscataway, NJ, USA), and data were visualized by ImageQuant TL 7.0 (GE Healthcare, Pittsburgh,
PA, USA).
3.4. Labeling Proteins with Biotin-Aldehyde
Rat plasma (2 mg/mL protein from each sample, final concentration) was incubated with 250
µ
M
biotin-aldehyde in 200 mM citric acid, 2 mM TCEP, pH 3, in the dark at 25
◦
C for 5 h. To quench the
labeling reaction, cysteamine (1 mM, final concentration) in 50 mM citric acid and 2 mM TCEP, pH 3,
was added to each sample and incubated for additional 3 h. Reaction solutions were stored at
−
80
◦
C
before analysis.
3.5. Western Blotting of Biotin-Labeled Proteins
Labeling reactions (35
µ
L) were mixed with 2
×
Laemmli loading buffer (35
µ
L) containing
350 mM dithiothreitol (DTT) and then boiled in water for 5 min. For each reaction, two aliquots (30
µ
L)
were loaded into two separate precast Tris-HCl gels (4%–15%, Bio-Rad) for sodium dodecyl sulfate

Molecules 2016,21, 1195 8 of 12
polyacrylamide gel electrophoresis (SDS-PAGE). One gel was used for Coomassie blue staining and
the other for Western blotting. For blotting, proteins from the gel were transferred onto an Immun-Blot
PVDF Membrane (0.2
µ
m, Bio-Rad) using transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol,
pH 8.3). The membranes were next blocked in 2% bovine serum albumin (BSA) in TBST (25 mM
Tris, 137 mM NaCl, 3 mM KCl, 0.1% Tween-20, pH 7.4) for 1 h. After blocking, the membrane was
washed with TBST for 3
×
10 min and incubated with 0.5
µ
g/mL streptavidin-horseradish peroxidase
(HRP) in 20 mL TBST for 1 h. The membrane was then washed again in TBST for 5
×
6 min and
incubated in PBS (68 mM NaCl, 1 mM KCl, 5 mM Na
2
HPO
4
, and 1 mM KH
2
PO
4
, pH 7.4) for 10 min.
After incubation, the buffer was discarded, and the chemiluminescence signal was developed by the
addition of 1 mL SuperSignal West Pico chemiluminescent substrate for 1 min. Chemiluminescence
was detected by FluorChem Imager SP (Alpha Innotech, San Leandro, CA, USA), and the image was
analyzed by ImageQuant TL 7.0 (GE Healthcare). The experiment was conducted in duplicate for
each sample.
3.6. Data Analysis for the Degree of Modification
Protein N-homocysteinylation in overall level and in each protein band
'
s level (See Figure 4) were
determined from the chemiluminescent intensity, which was normalized by using the Coomassie blue
staining intensity of overall protein bands in order to minimize the intensity variation from the protein
loading amount. Eventually, the normalized chemiluminescent intensity was compared to the control
group and the statistical analysis was performed using two-tailed t-test analysis by GraphPad Prism 6
(GraphPad Software Inc., La Jolla, CA, USA).
4. Conclusions
For the first time, global analysis of protein N-homocysteinylation was performed in the plasma
from rats with perturbed homocysteine metabolism. As expected, a general correlation between
“total homocysteine” and N-homocysteinylation was observed. Interestingly, more pronounced and
statistically significant changes were identified for some individual protein bands. While larger sets
of samples should be analyzed before a conclusive interpretation can be drawn, our method has
been shown to be suitable for quantitative analysis. Moreover, our results suggest that other factors
directly involved in homocysteine/homocysteine thiolactone metabolism, such as the activity of
cystathionine
β
-synthase (CBS) or paraoxonase 1 (PON1), may play more direct and pronounced roles
in N-homocysteinylation and facilitate the identification of N-Hcy proteins.
Supplementary Materials:
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
9/1195/s1.
Acknowledgments:
We thank Penny Beuning, Barry Karger and Alex Makriyannis at Northeastern University for
access to their instruments. This is a contribution number 1038 from the Barnett Institute. The work is supported
by the National Institutes of Health (GM101396 to Z.S.Z., HL061795, HG007690 and GM107618 to J.L.), the
American Heart Association Predoctoral Fellowship (09PRE2300071 to T.Z.) and the China Scholarship Council
Fund (201408330488 to J.M.).
Author Contributions:
J.L., S.D., R.C.D., Z.S.Z. and J.M. participated in the interpretation of the studies and the
revision of the manuscript; T.Z. drafted the manuscript, conducted the experiments and data analysis. L.P.P. and
D.E.H. prepared the plasma and serum samples and contributed to the data analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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