Identification of protein targets of 4-hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives

Article abstract of DOI:10.1021/tx700347w

Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress. Diffusible electrophilic α,β- unsaturated aldehydes, such as 4-hydroxynonenal (HNE), have been shown to modify proteins that mediate cell signaling (e.g., IKK and Keap1) and alter gene expression pathways responsible for inducing antioxidant genes, heat shock proteins, and the DNA damage response. To fully understand cellular responses to HNE, it is important to determine its protein targets in an unbiased fashion. This requires a strategy for detecting and isolating HNE-modified proteins regardless of the nature of the chemical linkage between HNE and its targets. Azido or alkynyl derivatives of HNE were synthesized and demonstrated to be equivalent to HNE in their ability to induce heme oxygenase induction and induce apoptosis in colon cancer (RKO) cells. Cells exposed to the tagged HNE derivatives were lysed and exposed to reagents to effect Staudinger ligation or copper-catalyzed Huisgen 1,3 dipolar cycloaddition reaction (click chemistry) to conjugate HNE-adducted proteins with biotin for subsequent affinity purification. Both strategies yielded efficient biotinylation of tagged HNE-protein conjugates, but click chemistry was found to be superior for the recovery of biotinylated proteins from streptavidin-coated beads. Biotinylated proteins were detected in lysates from RKO cell incubations with azido-HNE at concentrations as low as 1 μM. These proteins were affinity purified with streptavidin beads, and proteomic analysis was performed by linear ion trap mass spectrometry. Proteomic analysis revealed a dose-dependent increase in labeled proteins with increased sequence coverage at higher concentrations. Several proteins involved in stress signaling (heat shock proteins 70 and 90 and the 78-kDa glucose-regulated protein) were selectively adducted by azido- and alkynyl-HNE. The use of azido and alkynyl derivatives in conjunction with click chemistry appears to be a valuable approach for the identification of the protein targets of HNE.

Full text of DOI:10.1021/tx700347w

432
Chem. Res. Toxicol. 2008, 21, 432–444
Identification of Protein Targets of 4-Hydroxynonenal Using Click
Chemistry for ex Vivo Biotinylation of Azido and Alkynyl
Derivatives
Andrew Vila,†,4 Keri A. Tallman,‡,4 Aaron T. Jacobs,† Daniel C. Liebler,‡,§
Ned A. Porter,†,‡ and Lawrence J. Marnett*,†,‡,§
Departments of Biochemistry, Chemistry, and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in
Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt UniVersity, NashVille, Tennessee 37232
ReceiVed September 25, 2007
Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress.
Diffusible electrophilic R,ꢀ-unsaturated aldehydes, such as 4-hydroxynonenal (HNE), have been shown
to modify proteins that mediate cell signaling (e.g., IKK and Keap1) and alter gene expression pathways
responsible for inducing antioxidant genes, heat shock proteins, and the DNA damage response. To fully
understand cellular responses to HNE, it is important to determine its protein targets in an unbiased
fashion. This requires a strategy for detecting and isolating HNE-modified proteins regardless of the
nature of the chemical linkage between HNE and its targets. Azido or alkynyl derivatives of HNE were
synthesized and demonstrated to be equivalent to HNE in their ability to induce heme oxygenase induction
and induce apoptosis in colon cancer (RKO) cells. Cells exposed to the tagged HNE derivatives were
lysed and exposed to reagents to effect Staudinger ligation or copper-catalyzed Huisgen 1,3 dipolar
cycloaddition reaction (click chemistry) to conjugate HNE-adducted proteins with biotin for subsequent
affinity purification. Both strategies yielded efficient biotinylation of tagged HNE-protein conjugates,
but click chemistry was found to be superior for the recovery of biotinylated proteins from streptavidin-
coated beads. Biotinylated proteins were detected in lysates from RKO cell incubations with azido-HNE
at concentrations as low as 1 µM. These proteins were affinity purified with streptavidin beads, and
proteomic analysis was performed by linear ion trap mass spectrometry. Proteomic analysis revealed a
dose-dependent increase in labeled proteins with increased sequence coverage at higher concentrations.
Several proteins involved in stress signaling (heat shock proteins 70 and 90 and the 78-kDa glucose-
regulated protein) were selectively adducted by azido- and alkynyl-HNE. The use of azido and alkynyl
derivatives in conjunction with click chemistry appears to be a valuable approach for the identification
of the protein targets of HNE.
sion of several diseases including atherosclerosis, ischemia-
reperfusion injury, Parkinson’s disease, and Alzheimer’s disease
(4–8).
Introduction
Reactive oxygen species generated under conditions of
oxidative stress may initiate membrane lipid peroxidation (1).
Among the many products generated from lipid oxidation is
4-hydroxynonenal (HNE1) (2). This R,ꢀ-unsaturated aldehyde
covalently modifies DNA and protein resulting in genetic
mutations and altered cell signaling, respectively (3). HNE
modification of macromolecules may contribute to the progres-
Exposure of human colorectal cancer (RKO) cells to HNE
elicits gene expression responses such as the induction of various
antioxidant responsive, ER stress responsive, and heat shock
responsive transcripts (9). Extensive protein damage from HNE
treatment of RKO cells may account for the signaling responses
observed. Several studies have demonstrated that protein
modification by HNE or other electrophiles results in loss of
protein function and disruption of cellular signaling. HNE
modification of IκB kinase (IKK) (10), tubulin isoforms (11),
and Keap1 (12, 13) leads to altered function in signaling
pathways involved in NF-κB signaling, disruption of cytoskeletal
function, and protection against oxidative injury, respectively.
A comprehensive analysis of the proteins modified by HNE in
vivo is necessary to understand the role of oxidative stress on
cell signaling and disease pathology.
* To whom correspondence should be addressed. L. J. Marnett, Depart-
ment of Biochemistry, Vanderbilt University School of Medicine, Nashville,
TN 37232-0146. Tel: 615-343-7329. Fax: 615-343-7534. E-mail:
larry.marnett@vanderbilt.edu.
† Department of Biochemistry.
‡ Department of Chemistry.
§ Department of Pharmacology.
4 These authors contributed equally to this work.
1 Abbreviations: HNE, 4-hydroxy-2-nonenal; Az-HNE, azido-tagged
HNE; Al-HNE, alkynyl-tagged HNE; Az-Biot, biotin-conjugated azide; Al-
Biot, biotin-conjugated alkyne; BiotTPhPh, biotin-containing triphenyl
phosphine; SA, streptavidin; HRP-SA, streptavidin-conjugated horseradish
peroxidase; DMEM, Dulbecco’s modified Eagle’s medium; D-PBS, Dul-
becco’s phosphate buffered saline; TCEP, tris-(2-carboxyethyl)-phosphine-
HCl; BHA, butylated hydroxyanisole; MS/MS, tandem MS; LC-MS/MS,
liquid chromatography with electrospray ionization tandem MS; CHIPS,
complete hierarchical integration of protein searches.
The modification of proteins by HNE predominantly occurs
by Michael addition to nucleophilic amino acid residues His,
Cys, and Lys with a minor amount of Schiff base adducts to
Lys (14). The majority of previous studies identified HNE-
adducted proteins using Anti-HNE antibodies. Anti-HNE anti-
bodies directed against 4-HNE-sulfhydryl conjugates of keyhole
limpet hemocyanin (KLH) generated in rabbit hosts (15–18) or
10.1021/tx700347w CCC: $40.75
2008 American Chemical Society
Published on Web 01/31/2008

HNE Protein Targets
Scheme 1. Staudinger and Click Ligation Methods
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 433
proteins containing glycoconjugates of azido-sugars with a
biotin-containing triphenylphosphine (BiotTPhPh) using a modi-
fied Staudinger ligation (28, 29). The azido tag is stable in cells
and provides a chemical handle for specifically enriching protein
conjugates via biotin–avidin binding prior to LC-MS/MS
analysis. The second method uses click chemistry, or the
Huisgen 1,3-dipolar cycloaddition reaction, to conjugate azido
or alkynyl probes to terminal alkyne or azide reporter tags
(Biotin (30) or rhodamine (31)), respectively. Click chemistry
has been used for activity-based protein profiling (ABPP)
applications to identify serine esterase proteins in vivo (31–33).
Click chemistry also provides a high fidelity reaction between
protein targets in biological matrices and reporter groups similar
to the Staudinger ligation (34, 35). Also, azido or alkynyl probes
can be used for labeling cellular constituents followed by
coupling to alkynyl or azido reporter tags, respectively. Proteins
tagged with biotin from either of the above-mentioned methods
can be enriched through biotin–avidin binding prior to LC-MS/
MS analysis.
In the present study, we applied both the modified Staudinger
ligation and click chemistry approaches to label proteins in RKO
cells with HNE (1). Azido-tagged HNE (Az-HNE, 2) was used
to label proteins in intact RKO cells followed by conjugation
to BiotTPhPh (4) using the modified Staudinger ligation. For
click chemistry, both Az-HNE and alkynyl-tagged HNE (Al-
HNE, 3) were used to label proteins in intact cells followed by
conjugation with the appropriate biotin conjugated alkyne (Al-
Biot, 5) or azido (Az-Biot, 6), respectively. Biotin-conjugated
proteins were then enriched using streptavidin (SA) beads prior
to LC-MS/MS analysis. The results demonstrate that biotin
labeling of Az-HNE-adducted proteins via the modified Staudinger
ligation is efficient, but purification of biotin-labeled proteins
from background proteins using SA beads is difficult. The use
of click chemistry resulted in specific biotin labeling of Az-
HNE- and Al-HNE-adducted proteins and selective SA bead
purification of biotin-tagged proteins. Subsequent proteomic
analysis of these proteins revealed a broad spectrum of targets
for HNE.
antibodies specific for HNE-Michael adducts (10–13) have
been the most widely used for detecting HNE-protein adducts
in cells and tissues. The first reported HNE-protein adducts
were detected in a model of oxidative stress using carbon
tetrachloride (CCl4) treatment of isolated hepatocytes and rat
liver (18, 19). The protein adducts were detected with the anti-
HNE antibody specific for HNE-cysteine adducts. In a model
of alcoholic liver disease, the same antibody was used to detect
HNE-modified proteins on a Western blot of a 2D gel. The
HNE-modified proteins were identified as Hsp72 (inducible form
of Hsp70), Hsp90, and protein disulfide isomerase (PDI) by
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-MS) and liquid chromatography with
electrospray ionization (ESI) tandem MS (LC-MS/MS) (15–17).
Additionally, the same antibody was used to identify the
epithelial fatty acid binding protein (E-FABP) as a site of HNE
adduction in rat retinal homogenates (20). The identifications
of HNE-adducted protein in the above-mentioned studies are
limited because only HNE adducts on cysteine were detectable
with the antibody used.
Anti-HNE antibodies also show cross-reactivity with other
lipid electrophiles. A rabbit polyclonal antibody detected HNE-
modified proteins DRP-2, Hsp70, and R-enolase in a model of
familial amyotrophic lateral sclerosis (21). The specificity of
the antibody is for HNE- Michael adducts on His, Cys, and
Lys of proteins; however, 4-hydroxy-2-decenal and 4-hydroxy-
2-octenal can also be recognized (22). Another Michael adduct-
specific anti-HNE antibody (23) recognizes the reduced form
of the Lys-ONE Schiff base adduct, suggesting that this antibody
is also not specific for HNE-protein adducts (24). For a
comprehensive analysis of proteins modified by HNE, an
unbiased approach for identifying all types of HNE-protein
adducts is required. Also, a method for enriching HNE-adducted
proteins over nonadducted proteins prior to LC-MS/MS analysis
would enhance the overall identification of lower abundance
protein adducts.
Materials and Methods
Materials. Streptavidin conjugated to horseradish peroxidase
(HRP-SA), bovine serum albumin (BSA), sodium cyanoborohydride
(NaCNBH3), protease inhibitor cocktail (PIC) for mammalian cell
culture, dithiothreitol (DTT), and sodium ascorbate (Asc) were
purchased from Sigma-Aldrich (St. Louis, MO). Streptavidin
coupled to 6% cross-linked sepharose beads was from GE Health-
care (Picataway, NJ). Laemmli buffer, and silver staining kit were
from Biorad (Hercules, CA). Anhydrous CuSO4 was from Fisher
(Pittsburgh, PA). Gradient gels (4–20% polyacrylamide) were from
Invitrogen (Carlsbad, CA). Handee mini spin filters, M-Per cell
lysis buffer, West Pico ECL kit, and tris-(2-carboxyethyl)-
phosphine·HCl (TCEP) were purchased from Pierce (Rockford, Il).
Iodoacetamide was from Aldrich (Milwaukee, WI). Modified
trypsin, mass spectrometry grade, was from Promega (Madison,
WI). Western blotting was accomplished with Kodak Biomax light
film (Sigma) with ECL detection in an Electrophoresis Systems
Autoradiography Cassette (FisherBiotech; Wembley, WA).
Recently, biotin hydrazide has been used as a reporter tag
for proteins adducted by lipid oxidation products (25) and HNE
(26) in vitro and in vivo. Biotin hydrazide is highly specific for
free aldehyde groups on proteins (27) and several proteins
involved in the cellular stress response, lipotoxicity, and insulin
signaling were identified as being adducted by HNE. This
method has an advantage over immunochemical approaches in
that it recognizes HNE-protein adducts irrespective of the type
of adduct formed, and the biotin tag provides enrichment of
the labeled sample via biotin–avidin binding prior to LC-MS/
MS analysis. Although HNE-adducted proteins were identified
using this approach, oxidized proteins or proteins adducted by
other lipid aldehyde products were also identified. Therefore,
this method is not specific for proteins labeled with HNE.
General Methods. 1H and 13C NMR spectra were collected on
a 300 MHz NMR spectrometer. All reactions were carried out under
an atmosphere of argon. CH2Cl2was dried using a solvent purifica-
tion system. Commercial anhydrous DMSO, DMF, and CH3CN
were used as received. Purification by column chromatography was
carried out on silica gel, and TLC plates were visualized with
phosphomolybdic acid. HNE (1) and its analogues (2 and 3) were
synthesized following the same procedures; representative proce-
dures for HNE are presented as well as the characterization for all
Two recently developed methods for chemically tagging small
molecules in cells offer promise for identifying the protein
targets of reactive electrophiles such as HNE (Scheme 1). The
first method was developed for selective labeling of membrane

434 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Vila et al.
compounds. 6-Heptynal (36), 7 (37), 12 (38), and 6-azidohexanoic
acid (39) were synthesized by literature procedures.
2H, J ) 1.5, 7.2 Hz), 2.18 (dt, 2H, J ) 2.7, 6.9 Hz), 1.92 (t, 1H,
J ) 2.7 Hz), 1.60 (m, 4H), 1.27 (t, 3H, J ) 7.2 Hz), 0.89 (s, 9H),
0.04 (s, 3H), 0.01 (s, 3H); 13C NMR (CDCl3) δ 166.6, 150.5, 120.1,
84.0, 71.0, 68.6, 60.3, 36.1, 25.8, 23.5, 18.4, 18.2, 14.2, -4.6, -5.0;
HRMS (MALDI) calculated, 311.2042 (M + H); observed,
311.2046.
Synthesis of 7-Azidoheptanal. NaN3 (2.3 g, 0.035 mol) was
added to a solution of 7-bromoheptanol (3.6 mL, 0.023 mol) in
DMSO (75 mL). After stirring overnight, the reaction mixture was
diluted with H2O and extracted with ether. The organics were
washed with brine and dried over MgSO4. The product was isolated
as a pale yellow liquid (3.9 g, 100%) and used without purification.
1H NMR (CDCl3) δ 3.62 (t, 2H, J ) 6.6 Hz), 3.22 (t, 2H, J ) 6.9
Hz), 1.54 (m, 5H), 1.36 (m, 6H); 13C NMR (CDCl3) δ 62.8, 51.4,
32.5, 28.9, 28.7, 26.6, 25.6. Oxalyl chloride (2.7 mL, 0.030 mol)
was added to a solution of DMSO (4.1 mL, 0.058 mol) in CH2Cl2
(100 mL) at -78 °C. After 30 min, 7-azidoheptanol (3.9 g, 0.023
mol) was added and the reaction mixture stirred for 30 min,
followed by the addition of Et3N (16 mL, 0.11 mol). After stirring
overnight, the reaction mixture was diluted with H2O and extracted
with ether. The organics were washed with 10% HCl, saturated
NaHCO3, and brine, and dried over MgSO4. Purification by column
chromatography (10% EtOAc/hexanes) afforded the product as a
pale yellow liquid (2.9 g, 81%). Characterization of 7-azidoheptanal
9c (R ) (CH2)2N3): 1H NMR (CDCl3) δ 6.88 (dd, 1H, J ) 4.8,
15.6 Hz), 5.94 (dd, 1H, J ) 1.5, 15.6 Hz), 4.28 (m, 1H), 4.17 (dq,
2H, J ) 3.9, 7.2 Hz), 3.23 (t, 2H, J ) 6.9 Hz), 1.54 (m, 4H), 1.34
(m, 4H), 1.27 (t, 3H, J ) 7.2 Hz), 0.88 (s, 9H), 0.03 (s, 3H), 0.01
(s, 3H); 13C NMR (CDCl3) δ 166.7, 150.8, 119.9, 71.3, 60.3, 51.3,
37.1, 28.7, 26.7, 25.8, 24.2, 18.2, 14.2, -4.6, -5.0.
Synthesis of (E)-4-(tert-Butyldimethylsilyloxy)-2-nonen-1-ol
(10). DIBAL-H (20 mL of 1.4 M/toluene, 0.028 mol) was added
to a solution of 9 (3.2 g, 0.010 mol) in CH2Cl2 (50 mL) at 0 °C.
After 30 min, the reaction mixture was quenched with 10% HCl
and extracted with CH2Cl2. The organics were dried over MgSO4
and purified by column chromatography (10% EtOAc/hexanes). The
product (2.6 g) was isolated as a colorless liquid in 93% yield.
1
10a (R ) CH2CH3): H NMR (CDCl3) δ 5.69 (m, 2H), 4.09
1
was consistent with the literature (40). H NMR (CDCl3) δ 9.73
(m, 3H), 1.45 (m, 2H), 1.25 (m, 7H), 0.87 (s, 9H), 0.86 (t, 3H, J
) 6.9 Hz), 0.03 (s, 3H), 0.00 (s, 3H); 13C NMR (CDCl3) δ 135.5,
128.1, 72.7, 63.3, 38.2, 31.8, 25.9, 24.9, 22.6, 18.2, 14.1, -4.3,
-4.8; HRMS (MALDI) calculated, 273.2250 (M + H); observed,
273.2248.
(t, 1H, J ) 1.5 Hz), 3.23 (t, 2H, J ) 6.9 Hz), 2.41 (dt, 2H, J ) 1.8,
7.2 Hz), 1.58 (m, 4H), 1.34 (m, 4H); 13C NMR (CDCl3) δ 202.5,
51.3, 43.7, 28.6, 26.4, 21.8.
Synthesis of (E)-Ethyl 4-hydroxy-2-nonenoate (8). Heptalde-
hyde (2.9 mL, 0.021 mol), 6-heptynal, or 7-azidoheptanal and
piperidine (2.8 mL, 0.028 mol) were added to a solution of 7 (3.3
g, 0.014 mol) in CH3CN (70 mL). After stirring overnight, the
reaction mixture was diluted with saturated NH4Cl and extracted
with CH2Cl2. The organic layer was dried over MgSO4. The product
was purified by column chromatography (20% EtOAc/hexanes) and
isolated as a yellow liquid (2.6 g) in 91% yield.
10b (R ) C≡CH): 1H NMR (CDCl3) δ 5.69 (m, 2H), 4.12 (m,
3H), 2.16 (m, 2H), 1.91 (t, 1H, J ) 2.7 Hz), 1.53 (m, 5H), 0.86 (s,
9H), 0.02 (s, 3H), 0.00 (s, 3H); 13C NMR (CDCl3) δ 134.9, 128.6,
84.4, 72.2, 68.4, 63.1, 37.1, 25.9, 24.1, 18.4, 18.2, -4.3, -4.8;
HRMS (MALDI) calculated, 269.1937 (M + H); observed,
269.1940.
1
10c (R ) (CH2)2N3): H NMR (CDCl3) δ 5.67 (m, 2H), 4.11
8a (R ) CH2CH3): 1H NMR (CDCl3) δ 6.91 (dd, 1H, J ) 4.8,
15.6 Hz), 6.00 (dd, 1H, J ) 1.5, 15.6 Hz), 4.27 (m, 1H), 4.17 (q,
2H, J ) 7.2 Hz), 1.88 (d, 1H, J ) 4.8 Hz), 1.55 (m, 2H), 1.30 (m,
6H), 1.26 (t, 3H, J ) 7.2 Hz), 0.86 (m, 3H); 13C NMR (CDCl3) δ
166.6, 150.2, 120.1, 71.1, 60.4, 36.6, 31.6, 24.8, 22.5, 14.2, 14.0;
HRMS (MALDI) calculated, 201.1491 (M + H); observed,
201.1493.
(m, 3H), 3.23 (t, 2H, J ) 6.9 Hz), 1.56 (m, 2H), 1.47 (m, 2H),
1.32 (m, 5H), 0.87 (s, 9H), 0.02 (s, 3H), 0.00 (s, 3H); 13C NMR
(CDCl3) δ 135.1, 128.4, 72.5, 63.2, 51.4, 38.0, 28.8, 26.7, 25.9,
24.6, 18.2, -4.3, -4.8; HRMS (MALDI) calculated, 320.2346 (M
+ Li); observed, 320.2348.
Synthesis of (E)-4-(tert-Butyldimethylsilyloxy)-2-nonenal (11).
Oxalyl chloride (0.65 mL, 7.3 mmol) was added to a solution of
DMSO (1.0 mL, 14 mmol) in CH2Cl2 (28 mL) at -78 °C. After
20 min, a solution of 10 (1.5 g, 5.6 mmol) in CH2Cl2 (10 mL) was
added. After 30 min, Et3N (3.9 mL, 28 mmol) was added and the
reaction mixture allowed to warm to room temperature. After 3 h,
the reaction mixture was diluted with H2O and extracted with ether.
The organic layer was washed with 10% HCl, saturated NaHCO3,
and brine, and dried over MgSO4. Purification by column chro-
matography (10% EtOAc/hexanes) afforded the product (1.2 g,
82%) as a colorless liquid.
1
8b (R ) C≡CH): H NMR (CDCl3) δ 6.91 (dd, 1H, J ) 4.8,
15.6 Hz), 6.00 (dd, 1H, J ) 1.5, 15.6 Hz), 4.31 (m, 1H), 4.16 (q,
2H, J ) 7.2 Hz), 2.21 (m, 2H), 2.07 (d, 1H, J ) 4.8 Hz), 1.94 (t,
1H, J ) 2.7 Hz), 1.67 (m, 4H), 1.26 (t, 3H, J ) 7.2 Hz); 13C NMR
(CDCl3) δ 166.5, 149.8, 120.4, 83.8, 70.5, 68.8, 60.5, 35.4, 24.0,
18.2, 14.2; HRMS (MALDI) calculated, 197.1178 (M + H);
observed, 197.1181.
8c (R ) (CH2)2N3): 1H NMR (CDCl3) δ 6.90 (dd, 1H, J ) 5.1,
15.6 Hz), 5.99 (dd, 1H, J ) 1.5, 15.6 Hz), 4.28 (m, 1H), 4.16 (q,
2H, J ) 7.2 Hz), 3.23 (t, 2H, J ) 6.6 Hz), 2.00 (d, 1H, J ) 3.6
Hz), 1.57 (m, 4H), 1.36 (m, 4H), 1.26 (t, 3H, J ) 7.2 Hz); 13C
NMR (CDCl3) δ 166.5, 150.0, 120.2, 70.9, 60.5, 51.3, 36.3, 28.7,
26.5, 24.7, 14.2; HRMS (MALDI) calculated, 248.1586 (M + Li);
observed, 248.1578.
1
11a (R ) CH2CH3): H NMR (CDCl3) δ 9.54 (d, 1H, J ) 8.1
Hz), 6.77 (dd, 1H, J ) 4.5, 15.3 Hz), 6.23 (ddd, 1H, J ) 1.5, 7.8,
15.3 Hz), 4.38 (m, 1H), 1.54 (m, 2H), 1.28 (m, 6H), 0.88 (s, 9H),
0.85 (t, 3H, J ) 6.9 Hz), 0.04 (s, 3H), 0.00 (s, 3H); 13C NMR
(CDCl3) δ 193.7, 160.3, 130.6, 71.6, 37.1, 31.7, 25.8, 24.5, 22.5,
18.1, 14.0, -4.7, -4.9; HRMS (MALDI) calculated, 271.2093 (M
+ H); observed, 271.2097.
Synthesis of (E)-Ethyl 4-(tert-Butyldimethylsilyloxy)-2-non-
enoate (9). TBDMSCl (2.5 g, 0.017 mol) and imidazole (2.4 g,
0.035 mol) were added to a solution of 8 (2.6 g, 0.013 mol) in
DMF (30 mL). After stirring overnight, the reaction mixture was
diluted with H2O and extracted with ether. The organic layer was
washed with brine and dried over MgSO4. The product (3.2 g, 78%)
was isolated as a colorless liquid after column chromatography
(10% EtOAc/hexanes).
1
11b (R ) C≡CH): H NMR (CDCl3) δ 9.54 (d, 1H, J ) 8.1
Hz), 6.76 (dd, 1H, J ) 4.5, 15.6 Hz), 6.24 (ddd, 1H, J ) 1.5, 8.1,
15.6 Hz), 4.44 (m, 1H), 2.18 (dt, 2H, J ) 2.7, 6.9 Hz), 1.93 (t, 1H,
J ) 2.7 Hz), 1.69 (m, 2H), 1.56 (m, 2H), 0.88 (s, 9H), 0.04 (s,
3H), 0.00 (s, 3H); 13C NMR (CDCl3) δ 193.5, 159.6, 130.9, 83.8,
71.1, 68.8, 35.8, 25.7, 23.5, 18.3, 18.1, -4.7, -5.0; HRMS
(MALDI) calculated, 267.1780 (M + H); observed, 267.1777.
11c (R ) (CH2)2N3): 1H NMR (CDCl3) δ 9.55 (d, 1H, J ) 7.8
Hz), 6.76 (dd, 1H, J ) 4.8, 15.6 Hz), 6.24 (ddd, 1H, J ) 1.5, 7.8,
15.3 Hz), 4.41 (m, 1H), 3.24 (t, 2H, J ) 6.9 Hz), 1.57 (m, 4H),
1.36 (m, 4H), 0.88 (s, 9H), 0.04 (s, 3H), 0.01 (s, 3H); 13C NMR
(CDCl3) δ 193.6, 159.9, 130.8, 71.4, 51.3, 36.9, 28.7, 26.7, 25.7,
24.3, 18.1, -4.7, -4.9; HRMS (MALDI) calculated, 318.2189 (M
+ Li); observed, 318.2199.
9a (R ) CH2CH3): 1H NMR (CDCl3) δ 6.90 (dd, 1H, J ) 4.8,
15.6 Hz), 5.93 (dd, 1H, J ) 1.5, 15.3 Hz), 4.26 (m, 1H), 4.17 (dq,
2H, J ) 1.5, 7.2 Hz), 1.50 (m, 2H), 1.29–1.25 (m, 6H), 1.27 (t,
3H, J ) 6.9 Hz), 0.88 (s, 9H), 0.85 (t, 3H, J ) 6.9 Hz), 0.03 (s,
3H), 0.01 (s, 3H); 13C NMR (CDCl3) δ 166.8, 151.2, 119.6, 71.6,
60.3, 37.3, 31.8, 25.8, 24.5, 22.5, 18.2, 14.3, 14.0, -4.6, -4.9;
HRMS (MALDI) calculated, 315.2355 (M + H); observed,
315.2353.
1
9b (R ) C≡CH): H NMR (CDCl3) δ 6.88 (dd, 1H, J ) 4.8,
Synthesis of (E)-4-Hydroxy-2-nonenal (1). Aqueous HF (0.5
mL) was added to a solution of 11 (0.55 g, 2.0 mmol) in CH3CN
15.6 Hz), 5.95 (dd, 1H, J ) 1.8, 15.6 Hz), 4.33 (m, 1H), 4.17 (dq,

HNE Protein Targets
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 435
(10 mL). After 1 h, the reaction mixture was diluted with H2O and
extracted with CH2Cl2. The organic layer was dried over MgSO4.
The product (0.3 g) was isolated as a colorless liquid after column
chromatography (20% EtOAc/hexanes) in 88% yield.
with HNE, Az-, or Al-HNE at concentrations ranging from 1 to 80
µM in DMEM containing 10% fetal bovine serum (FBS) with
vehicle control (DMSO 0.5%, v/v) run alongside. Following 48 h
of incubation, media was aspirated, washed once in Dulbecco’s
phosphate buffered saline (D-PBS) and replaced with a solution of
1 µM calcein-AM (Molecular Probes) in D-PBS. After 30 min,
the fluorescence was measured using a SpectrasMax multiwell plate
reader (Molecular Devices) using an excitation of 494 nm, and an
emission of 517 nm.
Induction of Heme-oxygenase-1 (HO-1) expression was done by
incubating RKO cells with 5 and 25 µM HNE, Az-, or Al-HNE
for 8 h. Cells were lysed using M-Per buffer with 1% PIC, and
HO-1 expression was monitored by Western blotting using an anti
HO-1 antibody from BD Biosciences.
1
1 (R ) CH2CH3): H NMR (CDCl3) δ 9.53 (d, 1H, J ) 7.8
Hz), 6.80 (dd, 1H, J ) 4.8, 15.9 Hz), 6.27 (ddd, 1H, J ) 1.5, 8.1,
15.6 Hz), 4.39 (m, 1H), 2.35 (d, 1H, J ) 4.5 Hz), 1.59 (m, 2H),
1.34 (m, 6H), 0.86 (t, 3H, J ) 6.6 Hz); 13C NMR (CDCl3) δ 193.8,
159.4, 130.5, 71.0, 36.4, 31.5, 24.8, 22.5, 13.9; HRMS (MALDI)
calculated, 163.1310 (M + Li); observed, 163.1316.
2 (R ) C≡CH): 1H NMR (CDCl3) δ 9.54 (d, 1H, J ) 7.8 Hz),
6.81 (dd, 1H, J ) 4.5, 15.6 Hz), 6.29 (ddd, 1H, J ) 1.8, 8.1, 15.9
Hz), 4.46 (m, 1H), 2.35 (d, 1H, J ) 4.5 Hz), 2.23 (dt, 2H, J ) 2.7,
6.6 Hz), 1.95 (t, 1H, J ) 2.7 Hz), 1.72 (m, 4H); 13C NMR (CDCl3)
δ 193.6, 158.8, 130.7, 83.7, 70.4, 69.0, 35.1, 23.9, 18.1; HRMS
(MALDI) calculated, 159.0997 (M + Li); observed, 159.0989.
Click Chemistry of Peptide Adducts. Stock solutions of 5 (100
mM), 6 (100 mM), and ligand (100 mM) were prepared in DMSO.
Stock solutions of sodium ascorbate (500 mM) and CuSO4 (500
mM) were prepared in H2O. An aliquot (40 uL) of the peptide
adduction mixture described above was diluted with H2O (260 uL)
and CH3CN (100 uL) so that the HNE analogue concentration was
0.5 mM. The use of CH3CN as a cosolvent was necessary to ensure
complete dissolution of all reagents. The appropriate biotin reagent
(5 or 6, 20 µL, 5 mM), ascorbate (20 µL, 25 mM), ligand (20 µL,
5 mM), and CuSO4 (20 µL, 25 mM) were added to the peptide
adduct, vortexed, and allowed to react at room temperature, typically
for 1 h. The samples were analyzed by LC-MS as described above.
Adduction of Biotin to HNE-Modified Proteins in Intact
RKO Cells Using the Modified Staudinger Ligation or Click
Chemistry. RKO cells were seeded in 10 cm plates at 2.0 × 106
cells/plate in DMEM with FBS. The following morning, cells were
washed with 5 mL of D-PBS (2×) followed by the addition of
Az-HNE or Al-HNE in 5 mL of DMEM lacking FBS with DMSO
(1% v/v) or vehicle control (DMSO only) for 1 h at 37 °C. Cells
were then washed twice with D-PBS, scraped into 5 mL of PBS,
and collected after centrifugation (1,000 rpms, 5 min, 4 °C). For
the modified Staudinger ligation (Az-HNE concentrations 50, 100,
and 150 µM), cells were scraped washed again by centrifugation,
and lysed in M-Per lysis buffer containing 1% PIC for 20 min on
ice followed by sonication (10 pulses for 10 s, 35% power, 4 °C).
Freshly prepared NaCNBH3 (0.5 M in D-PBS) was added to the
cell lysates and incubated for 1 h to reduce Schiff base adducts
and reversible Michael adducts (41). BiotTPhPh (200 µM) was
added for 1 h, and the sample was quenched by boiling in laemmli
buffer with 5% ꢀ-mercaptoethanol, followed by Western blot
analysis to assess the level of biotin-tagging.
Click chemistry was performed with cellular lysates containing
1.0 mg/mL of protein with 0.6 mM of the appropriate biotin reagent
(Al-Biot or Az-Biot) (0.6 mM), TCEP (6 mM), ligand (13, 0.75
mM), and CuSO4 (6 mM). RKO cells treated with Az-HNE (1, 5,
10, 25, or 50 µM) or vehicle control were lysed as described above.
For assessing the time-dependence of Al-Biot conjugation with Az-
HNE, adducted proteins aliquots were removed from the incubation
at 0, 1.5, 3, and 6 h and added to an equal volume of laemmli
buffer containing 5% ꢀ-mercaptoethanol, 10 mM EDTA, and 100
µM BHA to quench the reaction. Samples were prepared for SA
beads as described below. RKO cells treated with Al-HNE (5, 10,
or 50 µM) were lysed with 100 mM HEPES at pH 7.5, 150 mM
NaCl, 0.1 mM EDTA, and 1% Igepal with 1% PIC, processed as
described above, and treated with Az-Biot for 2 h prior to SA bead
purification.
1
3 (R ) (CH2)2N3): H NMR (CDCl3) δ 9.53 (d, 1H, J ) 7.8
Hz), 6.80 (dd, 1H, J ) 4.8, 15.6 Hz), 6.27 (ddd, 1H, J ) 1.5, 7.8,
15.6 Hz), 4.40 (m, 1H), 3.24 (t, 2H, J ) 6.9 Hz), 2.41 (br s, 1H),
1.58 (m, 4H), 1.42 (m, 4H); 13C NMR (CDCl3) δ 193.7, 159.1,
130.6, 70.8, 51.2, 36.1, 28.6, 26.5, 24.7; HRMS (MALDI)
calculated, 204.1324 (M + Li); observed, 204.1333.
Synthesis of Biotin Alkyne (5). Oxalyl chloride (2.4 mL, 0.027
mol) was added to a solution of 5-hexynoic acid (2.0 mL, 0.018
mol) and DMF (0.30 mL, 0.0039 mol) in benzene (18 mL). After
2 h, the reaction mixture was concentrated. A solution of the acid
chloride in DMF (3.6 mL of 1.8 M, 6.5 mmol) was added to a
solution of 12 (2.0 g, 5.3 mmol) and Et3N (1.1 mL, 7.9 mmol) in
DMF (25 mL). After stirring overnight, the reaction mixture was
concentrated under high vacuum and purified by column chroma-
tography (10% MeOH/CH2Cl2). A solution of the product in MeOH
was passed through basic Dowex to remove triethylammonium salts
and isolated as an off-white powder (1.4 g, 57%). 1H NMR (MeOH-
d4) δ 4.49 (dd, 1H, J ) 4.8, 7.8 Hz), 4.30 (dd, 1H, J ) 4.5, 7.8
Hz), 3.61 (s, 4H), 3.54 (t, 4H, J ) 5.7 Hz), 3.36 (t, 4H, J ) 5.4
Hz), 3.20 (m, 1H), 2.92 (dd, 1H, J ) 5.1, 12.9 Hz), 2.70 (d, 1H, J
) 12.9 Hz), 2.31 (t, 2H, J ) 7.2 Hz), 2.21 (m, 5H), 1.79 (pentet,
2H, J ) 7.5 Hz), 1.64 (m, 4H), 1.43 (m, 2H); 13C NMR (MeOH-
d4) δ 176.1, 175.4, 166.0, 84.2, 71.2, 70.6, 70.5, 63.3, 61.6, 57.0,
54.8, 41.1, 40.2, 36.7, 35.8, 29.7, 29.5, 26.8, 25.9, 18.6; HRMS
(MALDI) calculated, 469.2485 (M + H); observed, 469.2488.
Synthesis of Biotin Azide (6). Biotin reagent 6 was synthesized
starting from 6-azidohexanoic acid as described above and isolated
as an off-white powder (1.4 g, 53%). 1H NMR (MeOH-d4) δ 4.49
(dd, 1H, J ) 4.8, 7.8 Hz), 4.30 (dd, 1H, J ) 4.2, 7.8 Hz), 3.61 (s,
4H), 3.54 (t, 4H, J ) 5.7 Hz), 3.35 (t, 4H, J ) 5.4 Hz), 3.29 (t,
2H, J ) 6.9 Hz), 3.20 (m, 1H), 2.92 (dd, 1H, J ) 5.1, 12.9 Hz),
2.70 (d, 1H, J ) 12.9 Hz), 2.21 (t, 4H, J ) 7.2 Hz), 1.65 (m, 8H),
1.42 (m, 4H); 13C NMR (MeOH-d4) δ 176.10, 176.05, 166.1, 71.3,
70.6, 63.3, 61.6, 57.0, 52.3, 41.1, 40.3, 36.8, 36.7, 29.8, 29.6, 29.5,
27.4, 26.8, 26.5; HRMS (MALDI) calculated, 514.2812 (M + H);
observed, 514.2817.
Peptide Adduction. Stock solutions of peptides (1 mM) were
prepared in phosphate buffer (50 mM, pH 7.4) and stock solutions
of HNE and analogues (51 mM) were prepared in DMSO. Initial
experiments were carried out with peptide (90 µL, 0.9 mM) and
HNE (10 µL, 5 mM) to identify the peptide adducts. For
experiments in which the reactivity of the analogues was compared
to HNE, samples were prepared with peptide (70 µL, 0.7 mM) and
an equimolar mixture of each HNE compound (10 µL of each HNE,
5 mM each). In all experiments, the samples were incubated at 37
°C, typically for 1 h. The reaction mixture was subsequently reduced
with NaBH4 (100 mM) then neutralized with 1 M HCl (10 µL) for
LC-MS analysis. The reaction mixture was analyzed on a Discovery
C18 column using a mobile phase consisting of A, 0.05% TFA in
H2O and B, 0.05% TFA in CH3CN. The unreacted peptide and
adducts were eluted with a gradient of 15% to 35% B over 20 min,
then to 90% B over 10 min, and back to 15% B over 5 min.
Biological Activity of HNE, Az-, and Al-HNE. For cell viability
assays, RKO cells were seeded in 96-well plates at a density of
7,500 cells per well. After adhering overnight, cells were treated
Purification of Biotin-Tagged Proteins from the Modified
Staudinger Ligation and Click Chemistry with Streptavidin
Beads. Purification of biotin-tagged proteins from the modified
Staudinger ligation was done with 30 µL of cell lysates (∼7.5 µg
of total protein) and 45 µL of SA beads (1:1 slurry in 0.1 M
ammonium bicarbonate) in Handee mini-spin columns with end-
over-end rotation for 1 h. After 1 h, the beads were pelleted at
6,500g for 5 min, and the supernatant was collected as the
breakthrough fraction (proteins that are not retained by streptavidin
beads). The beads were washed successively with ammonium
bicarbonate, 1 M NaCl with 1% Igepal, ammonium bicarbonate,

436 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Vila et al.
Scheme 2. Synthesis of HNE and Analogues
and distilled H2O. The first elution of biotin-tagged proteins was
accomplished with 70% CH3CN and 5% formic acid (FA). Another
wash with distilled H2O was done, and an equal volume of laemmli
buffer with (5% ꢀ-mercaptoethanol) was added to the SA beads
and boiled for 10 min for the second elution. Both Western blot
and silver stain analysis were carried out to determine where biotin-
tagged proteins and total proteins were eluting, respectively.
Biotin-tagged proteins from click chemistry reactions were
centrifuged (5,000g, 5 min, 4 °C) and recovered in the pellet,
washed in ice-cold methanol, and then resuspended by sonication
(5 s). Supernatant solutions contained undetectable levels of protein
(BCA assay) and biotin-conjugated protein (Western blotting) (data
not shown). The samples were then rotated end-over-end at 4 °C
for 10 min and centrifuged, and the supernatant was removed.
Following another methanol wash, protein samples were solubilized
in 0.1 M ammonium bicarbonate containing 0.2% SDS. Cell lysates
treated with control, 5, 10, and 50 µM for Az-HNE were incubated
with streptavidin-conjugated sepharose (SA) beads overnight in
Handee mini-spin columns on a shaker at 60 rpms. Each sample
contained a total of 150 µg of protein (0.5 mg/mL) with 75 µL of
1:1 slurry of SA beads in ammonium bicarbonate. Lysates from
Al-HNE-treated (5, 10, and 50 µM) RKO cells contained 2 mg of
protein (1.0 mg/mL) and were incubated with a total of 1 mL of
SA beads in 1.5 mL tubes for 2 h. Also, click chemistry reactions
with Az-HNE-adducted proteins from RKO cells were done using
2 mg of cell lysate for comparison with Al-HNE. The 1.5 mL tubes
were centrifuged (6,500g, 5 min), and the breakthrough fraction
was collected. More stringent wash steps were used in the
experiments with 2 mg of cellular lysate. The beads were washed
successively with 1% SDS, 4 M urea, 1 M NaCl, ammonium
bicarbonate, and distilled water. The beads were incubated for 30
min with 1% SDS and 4 M urea, and for 10 min with 1 M NaCl
with end-over-end rotation prior to pelleting the beads. Biotin-tagged
proteins were eluted as described above.
Proteomic Analysis of Captured Proteins. Biotin-tagged
proteins from Coomassie stained gels were subjected to in-gel
digestion, followed by peptide extraction, and proteomic analysis
by LC-MS/MS using an established procedure (42). Biotin-tagged
proteins purified with SA beads were run on a gradient gel (4–20%
acrylamide) and visualized with colloidal Coomassie blue (Invit-
rogen). Visible bands were excised by cutting the entire molecular
weight range into 10 fractions with industrial razor blades (VWR),
which were further sliced into ∼1 mm cubes, and placed into a 1.5
mL eppendorf tube. The cubes were washed once with 100 µL of
ammonium bicarbonate and centrifuged, and another 100 µL of
ammonium bicarbonate was added. DTT was added to a final
concentration of 3 mM, and the cubes were incubated for 15 min
at 50 °C followed by incubation with iodoacetamide (6.25 mM final
conc) for 15 min. The samples were then equilibrated with 50:50
CH3CN/ammonium bicarbonate for 15 min to remove the blue stain.
The gel cubes were then dehydrated with 100% CH3CN followed
by desiccation in a vacuum centrifuge. The dehydrated gel slices
were reswelled with 25 µL of 0.01 µg/µL modified trypsin in 25
mM ammonium bicarbonate and incubated overnight at 37 °C.
Peptides were extracted with 50 µL of 60% CH3CN, 1% FA (2×),
evaporated in vacuo, resuspended in distilled water with 1% FA,
and then desalted using Zip-Tips (Millipore C18, P10). The peptide
eluant from the Zip-Tips was evaporated, and 50 µL of distilled
water with 0.1% FA was added followed by LC-MS/MS analysis.
equilibration to 98% A. MS/MS spectra were acquired using one
data-dependent scan from the most intense precursor ion in the full
scan mode. The MS/MS spectra were matched to human database
sequences with TurboSequest. S-carbomethylation of Cys (+57
amu) and oxidation of Met (+16 amu) were specified as dynamic
modifications. Sequest output files were filtered with the software
database system CHIPS (Complete Hierarchical Integration of
Protein Searches). Sequence-spectrum assignments were accepted
on the basis of the following filtering criteria: (1) all peptide
sequence assignments were required to result from fully tryptic
cleavages, (2) singly, doubly, and triply charged ions were accepted
if their XCorr scores were greater than 2, 2.5, and 3, respectively,
and (3) all putative matches were confirmed by visual inspection
of the spectra. Identifications were accepted only for proteins with
3 unique peptide identifications matching high quality MS/MS
spectra. The lists of proteins from vehicle control, 5, 10, and 50
µM Az- and Al-HNE treated cells were compared in CHIPS.
SDS-PAGE and Western Blotting with ECL Detection. Tris-
glycine polyacrylamide gels (4–20%) were used for separating
proteins. Samples in laemmli sample buffer with 5% ꢀ-mercapto-
ethanol were run on the gels for 1 h at 150 V. The silver stain plus
kit (Biorad) was used to fix and stain gels for the visualization of
total protein. Visualization of biotin-tagged protein was done by
Western blot analysis. Proteins separated on gradient gels were
transferred to a 0.2 µm nitrocellulose membrane for 1 h in
SDS-PAGE transfer buffer (1× running buffer with 20% MeOH)
at 100 V. The membranes were blocked with PBS with Tween20
(0.1% v/v) (PBS-T) for 1 h and then blotted with HRP-SA (100
ng/mL) for an additional hour. The membranes were then washed
in PBS-T, and biotin-tagged proteins recognized by HRP-SA were
detected on BioMax light film using the SuperSignal West Pico
Chemiluminescent Substrate.
Results
Synthesis of HNE Analogues and Biotin Tags. HNE and
its analogues were synthesized in a similar manner (Scheme
2), modified from a previously reported procedure (37). Sulfinate
7 was coupled with the appropriate aldehyde to yield the R,ꢀ-
unsaturated esters (8) in good yields. Subsequent protection of
the alcohol as the silyl ether was necessary to enable facile
reduction of the ester to the alcohol (10). The ester was
selectively reduced to the alcohol using DIBAL-H, whereas
LiAlH4 also reduced the olefin. Alcohol 10 was oxidized to the
aldehyde (11) under Swern conditions and finally deprotected
to yield HNE and its analogues (1–3). Several common silyl
deprotection strategies were explored, such as TBAF and
pyridine-HF, but we found significant decomposition of the
product under these conditions. Optimum yields were obtained
when the mild HF conditions were used.
The samples were analyzed on a Thermo LTQ linear ion trap
instrument equipped with a Thermo microelectrospray source, and
a Thermo Surveyor pump and autosampler. LC-MS/MS analyses
were done by reverse phase chromatography on an 11 cm fused
silica capillary column (100 µm i.d.) packed with Jupiter C-18 (5
µm) resin set to a flow rate of 700 nL/min. The mobile phase
consisted of 0.1% formic acid in either HPLC grade H2O (A) or
CH3CN (B), and sample runs were 95 min in length. The peptide
samples were injected onto the column at 98% A, which was held
for 15 min, followed by a linear gradient from 98% A to 75% A
for 50 min. A linear gradient to 10% A at 65 min was done followed
by isocratic conditions of 10% A to 74 min followed by re-
BiotTPhPh was synthesized according to previous procedures
(28). The biotin reagents for click chemistry were designed to
contain either an azide or alkyne functionality for coupling to
2 and 3, respectively, under click conditions. In addition, the

HNE Protein Targets
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 437
Figure 1. Comparison of HNE, Az-HNE, and Al-HNE towards cell toxicity and induction of heme-oxygenase-1 (HO-1) expression. Cell viability
assays were done with cells seeded in 96-well plates at a density of 7.5 × 103 cells per well. After adhering overnight, cells were treated with HNE,
Az-HNE, or Al-HNE (1–80 µM) in DMEM containing 10% fetal bovine serum. After 48 h, cell viability was determined using calcein-AM. Data
in panel A are represented as mean % control fluorescence (mean ratio of arbitrary fluorescence units of treated samples to DMSO-treated samples
× 100). The error bars represent standard deviations (n ) 8). Panel B shows a Western blot analysis of HO-1 induction in RKO cells. Cells were
treated with 5 and 25 µM HNE, Az-HNE, or Al-HNE for 8 h. Cell were then lysed, and Western blot analysis was done using an anti HO-1
antibody.
Scheme 3. Synthesis of Biotinylating Reagents
Scheme 4. Click Chemistry of Peptide Adduct with Biotin
Tag
biotin tag contains an ethylene glycol linker to increase water
solubility. Biotin was coupled to the linker to yield 12 as
previously reported (38). Reaction of 12 with 5-hexynoyl
chloride or 6-azidohexanoyl chloride produced the desired tags
5 and 6, respectively.
Peptide Adduction with HNE and Analogues. The HNE
analogues were designed to minimize structural variations
compared to those of native HNE that may result in a change
in reactivity. In order to compare the reactivity of 2 and 3
relative to 1, the adduction to several peptides was studied. Three
peptides were chosen that contained His, Lys, or Cys, major
sites of HNE adduction: Angiotensin II (Asp-Arg-Val-Tyr-Ile-
His-Pro-Phe), Thymic humoral gamma 2 factor (Leu-Glu-Asp-
Gly Pro-Lys-Phe-Leu), and ANP (Ser-Leu-Arg-Arg-Ser-Ser-
Cys-Phe-Gly Gly Arg). Each peptide (1 mM) was incubated in
the presence of 1, 2, or 3 (5 mM) at 37 °C, typically for 1 h.
The reaction mixture was subsequently reduced with NaBH4,
neutralized, and analyzed by LC-MS for adduct formation. In
all cases, reduced Michael and Schiff base adducts were
observed, as well as a minor amount of a mixture of both
adducts (see Supporting Information, Figure S1).
These qualitative experiments indicated that modification of
the HNE structure did not impact the formation of Schiff base
and Michael adducts. However, we were also interested in the
relative reactivity of analogues 2 and 3 compared to 1. Each
peptide (0.7 mM) was incubated in the presence of an equimolar
mixture of 1–3 (5 mM each) as described above, and aliquots
were taken every hour. The samples were subsequently analyzed
by LC-MS, and the amount of each HNE adduct was measured.
For the peptides containing His and Lys, there was no significant
difference between the relative amounts of adducts formed with
each of the HNE analogues compared to native HNE. However,
when the peptide contained Cys, there was approximately 50%
less adduct in the presence of Az-HNE (Figure S2). The reason
for the less efficient reaction of the Cys-containing peptide with
Az-HNE is unknown, but it may be from thiol reduction of the
azide moiety.
were carried out with an equimolar mixture of the peptides in
the presence of each HNE compound. As expected, the Lys-
and Cys-containing peptides were considerably more reactive
than the His-containing peptide. Consistent with the previous
experiment, analogues 2 and 3 exhibited reactivity similar to
that of HNE, except when a Cys was present in the peptide. As
observed in the HNE mixture experiments, incubation of Cys
in the presence of Az-HNE generated approximately 50% less
adduct (Figure S3).
Comparison of the Biological Activity of HNE and
Analogues. The structural similarity of HNE to its analogues
was also tested by comparing their relative toxicity and ability
to induce heme-oxygenase-1 (HO-1) expression. RKO cells were
treated with HNE, Az-, or Al-HNE, and cellular viability was
determined after 48 h of incubation. As shown in Figure 1A,
the cellular viability for HNE and its analogues are very similar.
Induction of HO-1 expression with exposure of RKO cells to
25 µM HNE, Az-, or Al-HNE for 8 h was similar (Figure 1B).
Therefore, Az- and Al-HNE are suitable analogues of HNE to
probe the reactivity with proteins in vivo.
Click Chemistry Optimization of HNE-Adducted Peptides.
Peptides adducted with either Al- or Az-HNE were subsequently
tagged with biotin for affinity purification utilizing click
chemistry (Scheme 4). The click reaction employs a Cu(I)
catalyzed cyclization of an azide with an alkyne to form a stable
triazole. There are a variety of reaction conditions published
To further explore the reactivity of the HNE analogues and
more closely mimic biological conditions, adduction experiments

438 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Vila et al.
Figure 2. Incubation of Angiotensin with (A) Al-HNE and Az-biotin, and (B) Az-HNE and Al-biotin. From top to bottom are the unreacted
peptide, peptide Schiff base and Michael adducts, and triazole. Angiotensin (1 mM) was incubated with Al- or Az-HNE (5 mM) at 37 °C for 1 h,
then with Az- or Al-biotin (2–5 equiv), Asc (50 equiv), 13 (10 equiv), and CuSO4 (50 equiv) for 1 h.
for this particular reaction; therefore, conditions were optimized
for our particular reagents. The most striking differences are
the CuSO4 reducing agent and the choice of amine to stabilize
the in situ formed Cu(I) species. We explored the use of tris(2-
carboxyethyl)phosphine or Asc as the reducing agent, and ligand
13 or diisopropylethylamine (DIPEA) as the stabilizer. The
peptide adduct was reacted with the appropriate biotin tag (5
or 6) under a variety of conditions, and the progress of the
reaction was monitored by LC/MS.
From the studies on the click chemistry of the peptide adducts,
we found that Asc was superior to TCEP as the CuSO4 reducing
agent. When TCEP was used, there was negligible triazole
formation, as detected by LC/MS. Increasing the reaction time
and equivalents of CuSO4/TCEP had little effect on the reaction.
In addition, the use of 13 or DIPEA did not increase the yield
of triazole. However, when Asc was used as the reducing agent
in conjunction with 13, complete triazole formation was
observed within 1 h. These latter reaction conditions were tested
for all peptides mentioned above that had been adducted with
either Al- or Az-HNE and subsequently reacted with Az- or
Al-biotin, respectively. All adducted peptides were quantitatively
converted to their biotin-tagged counterparts within 1 h. Shown
in Figure 2 is a representative analysis of the peptide adduction
and the subsequent click chemistry with the biotin tags to form
triazole.
Figure 3. Staudinger ligation for biotin-labeling of Az-HNE adducted
proteins in RKO cells. Intact RKO cells were incubated with Az-HNE
(50, 100, and 150 µM) and vehicle control (DMSO) for 1 h. The cells
were lysed as described in Materials and Methods and then incubated
with 200 µM BiotTPhPh for 1 h. The degree of biotin labeling was
determined by Western blotting using HRP-SA. Protein load was 2.5
µg per lane. S, biotin-labeled standards; B, RKO cells incubated with
no DMSO; 0, RKO cells incubated with DMSO (1% v/v); 50, 100,
and 150 are the concentrations of Az-HNE added to the cells in DMSO
(1% v/v).
efficiently labeled with BiotTPhPh after 1 h of incubation
(Figure 3). Cells exposed to 50, 100, and 150 µM Az-HNE were
efficiently labeled with biotin as judged by the Western blot
signal using HRP-SA, whereas there was no detectable biotin
signal for cell samples treated with vehicle control.
Biotin Labeling of Az-HNE-Adducted Protein in RKO
Cells with the Staudinger Ligation. Initial studies were aimed
at biotin labeling Az-HNE-adducted proteins using the modified
Staudinger ligation. RKO cells labeled with Az-HNE were

HNE Protein Targets
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 439
Figure 4. SA bead purification of biotin-tagged proteins produced from
the Staudinger ligation. Cellular lysates (lane 1) containing biotin-tagged
proteins (30 µL, ∼7.5 µg of protein) were incubated with SA beads (30
µL; 1:1 in 0.1 M ammonium bicarbonate) for 1 h, then the beads and the
breakthrough fraction (lane 2) were recovered by centrifugation. The beads
were washed successively with ammonium bicarbonate (lane 3), 1 M NaCl
with 1% Igepal (lane 4), ammonium bicarbonate (lane 5), and distilled
H2O (lane 6). Protein elution was done in two steps. lane 7 represents
elution with 70% CH3CN and 5% FA. lane 8 represents a wash with
distilled H2O, and lane 9 represents elution by boiling in laemmli buffer
containing 5% ꢀ-mercaptoethanol. Panels A and C are the vehicle control
samples, and panels B and D are 50 µM Az-HNE samples. Western blotting
was performed in panels A and B, and silver stain analysis was done in
panels C and D. S, biotin labeled standards.
Figure 5. Time dependence of click chemistry for labeling Az-HNE
adducted proteins in RKO cells. Intact RKO cells were incubated with
Az-HNE (1, 5, 10, 25, and 50 µM) and vehicle control (DMSO) for
1 h. The cells were lysed as described in Materials and Methods and
then incubated with biotin linker alkyne (600 µM), TCEP (6.0 mM),
ligand (13, 0.75 mM), and CuSO4 (6.0 mM) for 0, 1.5, 3, and 6 h. The
degree of biotin labeling with click chemistry is determined by Western
blotting using HRP-SA. Protein load was 2.5 µg/lane. The biotin
labeling is shown for the control (panel A), 1 µM Az-HNE (panel B),
5 and 10 µM Az-HNE (panel C), and 25 and 50 µM (panel D).
Affinity Purification of Proteins Biotin-Tagged with the
Modified Staudinger Ligation Using SA Beads. Purification
of biotin-tagged proteins using SA beads was attempted prior
to subsequent proteomic analysis. Western blot analysis dem-
onstrated that biotin-tagged proteins readily bound to SA beads
(lane 2, Figure 4B). Also, biotin-tagged proteins were retained
on the SA beads in subsequent wash steps with significant
elution of non-adducted proteins (lanes 3–6, Figure 4A and B).
There was no elution of biotin-tagged proteins from SA beads
using 70% CH3CN and 5% FA (lane 7, Figure 4B); however,
boiling the beads in laemmli buffer resulted in significant elution
of biotin-tagged proteins (lane 9, Figure 4B). Unfortunately,
detection of total proteins by silver stain analysis showed that
there was significant contamination of nonadducted proteins with
biotin-tagged proteins (compare lane 9 of Figure 4C and D).
Therefore, biotin-tagged proteins from the modified Staudinger
ligation were not affinity-purified with SA beads, and subsequent
proteomic analysis was not performed.
Click Chemistry of Cellular Proteins Labeled with Az-
HNE in RKO Cells. Click chemistry was applied to conjugate
HNE-adducted proteins with biotin as an alternative to the
Staudinger ligation. Intact RKO cells were treated with Az-
HNE (1, 5, 10, 25, and 50 µM), then the time dependence of
Al-Biot conjugation to Az-HNE adducted proteins from cellular
lysates was tested using click chemistry. Lysates from cells
treated with 50 and 25 µM Az-HNE reached maximal biotin
labeling after 90 min of incubation as judged by HRP-SA
detection by Western blotting (Figure 5D). Comparatively,
lysates from cells treated with 5 and 10 µM Az-HNE needed
3–6 h of incubation for maximal labeling (Figure 5C). There
was no detection of biotin-tagged proteins in DMSO control
samples up to 3 h of reaction (Figure 5A). However, there was
a barely detectable signal in the DMSO controls after 6 h (Figure
5A). Biotin-tagged proteins from cells treated with as low as 1
µM Az-HNE were detected with a significantly higher signal
compared to that of DMSO controls after 6 h of reaction (Figure
5B vs A, respectively). It is interesting to note that the click
chemistry reaction occurred in cell lysates with TCEP as the
reductant, whereas the same reaction did not occur with peptide
adducts. This is puzzling; however, different reaction conditions
(i.e., Az-HNE-adducted peptides in aqueous solution vs Az-
HNE-adducted proteins in crude cell lysates) may have con-
tributed to this difference. Click chemistry reactions performed
with cell lysates in the presence of ascorbate were complete
after ∼5 min (data not shown), as compared to ∼90 min for
TCEP. The much higher rate for the click chemistry reaction
with ascorbate compared to that for TCEP is consistent with
the model peptide studies. However, biotin-tagged proteins were
not affinity purified from nonmodified proteins with SA beads
when ascorbate was used as the reductant (data not shown). As
shown below, using TCEP as the reductant resulted in specific
recovery of biotin-tagged proteins. Therefore, TCEP was used
in further experiments.

440 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Vila et al.
32, 41, 107, and 118, respectively. Proteins unique to the 5,
10, and 50 µM Az-HNE-treated cells compared to vehicle
controls were 12, 58, and 91, respectively. A complete list of
unique protein targets from Az-HNE treatment is displayed in
Supporting Information (Table S1). The number of proteins
identified corresponded to the concentration of Az-HNE used
(i.e., increase in protein hits with increasing Az-HNE concentra-
tion), suggesting that the method is specific for HNE adduction
of proteins. There were 39 common proteins identified between
50 and 10 µM Az-HNE-treated samples (Table S1). Also, 8
out of 12 proteins identified in the 5 µM sample were also
present in the 50 µM sample (Table S1). Overall, the unique
proteins found in the 50 µM Az-HNE-treated samples had better
quality MS/MS spectra, more distinct peptides identified, and
greater sequence coverage compared to the 10 and 5 µM treated
samples (data not shown). Table 1 shows a select list of stress
signaling proteins corresponding to the heat shock response and
ER stress response such as Hsp70 and Hsp90, 78-kDa glucose
regulated protein (GRP-78), respectively, as well as proteins
involved in redox regulation, xenobiotic metabolism, and protein
folding (glutathione-S-transferase Pi, peroxiredoxin-1, and the
60 kDa mitochondrial heat shock protein, respectively). Hsp70,
Hsp90, and protein disulfide isomerase isoform A3 (PDIA3)
were identified previously as being modified by HNE in vivo
(15–17). Table 2 shows the percent sequence coverage for the
select list of proteins in correlation with the increase in Az-
HNE concentration. GRP-78, peroxiredoxin-1, 60 kDa mito-
chondrial heat shock protein, and stress-70 protein are identified
in the lowest Az-HNE concentration used (5 µM) and may
represent significant targets of HNE adduction. Also, the percent
sequence coverage for these proteins increases dramatically from
5 to 50 µM Az-HNE.
Figure 6. SA bead purification of Az-HNE adducted protein conjugated
to biotin. Cellular lysates (lane 1) containing biotin-tagged proteins
(∼150 µg) were incubated with SA beads (75 µL; 1:1 in ammonium
bicarbonate) overnight, and then the beads were recovered in the
morning, and the breakthrough fraction (lane 2) was collected. The
beads were washed successively with ammonium bicarbonate (lane 3),
2 times with 1 M NaCl with 1% Igepal (lane 4 and 5), ammonium
bicarbonate (lane 6), and distilled H2O (lane 7). Protein elution was
done in two steps. Lane 8 represents elution with 70% CH3CN and
5% FA, and lane 9 represents elution by boiling in laemmli buffer
containing 5% ꢀ-mercaptoethanol. Panels A and C are the vehicle
control samples, and panels B and D are the 50 µM Az-HNE samples.
Western blotting was performed in panels A and B, and silver stain
analysis was done in panels C and D. S, biotin-labeled standard curve.
Click Chemistry and Proteomic Analysis of Biotin
Conjugated to Al-HNE-Adducted Protein. To confirm the
findings with Az-HNE, we repeated the same procedure with
Al-HNE. In these experiments, we used 2 mg of cell lysates to
obtain better MS/MS spectra and identification of proteins. We
first used the same lysis buffer for Al-HNE-treated cells as with
Az-HNE-treated cells. However, the conjugation of Az-Biot to
Al-HNE-adducted proteins was negligible in the 5 and 10 µM
Al-HNE-treated samples after several hours of click chemistry
(data not shown). Therefore, we switched to using 100 mM
HEPES at pH 7.5, 150 mM NaCl, 0.1 mM EDTA, and 1%
Igepal as the lysis buffer. This resulted in efficient labeling of
Al-HNE-adducted proteins with Az-Biot as shown in Figure 7
(lane 1; panel A for 0 and 5 µM, and panel B for 10 and 50
µM Al-HNE). There was an increase in biotin labeling of Al-
HNE-adducted proteins from 5 to 50 µM Al-HNE as demon-
strated with Az-HNE labeling (Figure 5). Affinity purification
of biotin-tagged proteins from lysates of cells treated with 5,
10, and 50 µM Al-HNE is shown in Figure 7. There is efficient
capture of biotin-tagged proteins with the 5 and 10 µM Al-
HNE-treated sample (lanes 2–5, Figure 7A and B), with some
flow through of biotin-tagged proteins in the 50 µM sample.
Elution of biotin-tagged proteins from 5, 10, and 50 µM Al-
HNE-treated samples is shown in lane 6 of Figures 7A and B.
Proteomic Analysis of Al-HNE Modified Proteins. The total
numbers of proteins identified from the control, 5, 10, and 50
µM Al-HNE-treated RKO cells were 50, 276, 456, and 538. In
comparison, 21, 171, 67, and 322 proteins were identified as
adducted in the control, 5, 10, and 50 µM Az-HNE-treated cells,
respectively, using 2 mg of cell lysate for click chemistry and
SA bead purification. A complete list of proteins for Al-HNE
and Az-HNE from 2 mg of cell lysates is shown in Supporting
Streptavidin Purification of Proteins Biotin-Tagged with
Click Chemistry. Biotin-tagged proteins from lysates of cells
treated with Az-HNE (5, 10, or 50 µM) were affinity purified
with SA beads. Western blot analysis demonstrates that biotin-
tagged proteins from lysates of cells treated with 50 µM Az-
HNE were efficiently captured with SA beads (lane 2, Figure
6B) with no detectable proteins in the elution fractions (lanes
3–7, Figure 6B). Figure 6B shows the elution of biotin-tagged
proteins using 70% CH3CN and 5% FA (lane 8) and boiling in
laemmli buffer (lane 9). As shown in Figure 6A, DMSO controls
had a low biotin signal in cell lysates (lane 1), breakthrough
(lane 2), wash (lanes 3–7), or elution (lanes 8–9) fractions. The
same fractions analyzed by Western blotting were tested by
silver stain analysis for total protein detection. The lysate and
flow through lanes (lanes 1 and 2, Figure 6C and D) had similar
levels of total protein for both the control and 50 µM Az-HNE
treated samples, and the wash steps had higher levels of total
protein in the sample for 50 µM Az-HNE-treated samples
compared to that in control samples, respectively (lanes 3–5,
Figure 6D and C, respectively). In the CH3CN elution step,
significantly more proteins were eluted from SA beads incubated
with the 50 µM Az-HNE treated sample versus controls (lane
8, Figure 6D and C, respectively). Similar protein bands were
observed in the eluant from boiling SA beads for both the control
and 50 µM Az-HNE-treated samples (lane 9, Figure 6C and D,
respectively). Therefore, biotin-tagged proteins were selectively
enriched relative to nontagged proteins by elution from SA beads
with 70% CH3CN and 5% FA.
Proteomic Analysis of Proteins Isolated with SA Beads.
The total number of proteins identified from cells treated with
vehicle control, 5, 10, and 50 µM Az-HNE-treated samples were

HNE Protein Targets
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 441
Table 1. Select Proteins Identified by LC-MS/MS Analysis of Az-HNE Adducted Proteinsa
protein
Swiss-Prot ID
functionb
protein folding
protein folding
protein folding
protein folding
protein folding
localizationc
heat shock 70 kDa protein 1
heat shock 70 kDa protein 1L
heat shock 70 kDa protein 4
heat shock 90 kDa R-2
78 kDa glucose regulated
protein
P08107
P34931
O14992
P07900
P11021
Cyt
Cyt
Cyt
Cyt
ER
protein disulfide isomerase A3
glutathione-S-transferase-Pi
peroxiredoxin-1
60 kDa mitochondrial heat
shock protein
P30101
P09211
Q06830
P10809
protein folding
Cyt
Cyt
Cyt
MT
xenobiotic metabolism
redox regulation
protein trafficking, protein
folding
stress-70 protein
P38646
cell proliferation
MT
a Proteins are selected from experiments with intact RKO cells treated with 50 µM Az-HNE. Total protein content of the cell lysates was 150 µg
prior to SA bead purification. b Protein function is taken from ingenuity analysis (http://www.ingeuity.com). c Protein localization is taken from the
protein information resource Web site (http://pir.georgetown.edu). Cyt, cytoplasm; ER, endoplasmic reticulum; MT, mitochondria.
Table 2. Percent Coverage of Select Proteins Adducted by
Az-HNE in RKO Cellsa
Table 3. Select Proteins Adducted by Both Al-HNEa and
Az-HNEb
protein
0 µM 5 µM 10 µM 50 µM
protein
0 µM 5 µM 10 µM 50 µM
heat shock 70 kDa protein 1
heat shock 70 kDa protein 1L
heat shock 70 kDa protein 4
heat shock 90 kDa R-2
78 kDa glucose regulated protein
protein disulfide isomerase A3
glutathione-S-transferase-Pi
peroxiredoxin-1
9b
7
8
6
24
7
36
39
22
25
heat shock 70 kDa protein 1
heat shock 70 kDa protein 1L
heat shock 70 kDa protein 4
heat shock 90 kDa R-2
78 kDa glucose regulated protein
protein disulfide isomerase A3
glutathione-S-transferase-Pi
peroxiredoxin-1
–a, –b 17, 7 15, – 21, 12
–, –
–, –
–, 3
9, 4
13, – 15, 9
6
3
23
27, 9 33, 3 37, 18
11, 4 14, 4 16, 6
9
13, 10 39, 27 34, 6 46, 33
–, –
–, –
18, 23 19, 7 31, 26
32, 18 48, 18 58, 5
23
37
10
10
16
8
10
14, – 34, 11 28, 11 51, 13
40, 23 44, 7 48, 47
12, 3 37, 23 42, 12 42, 30
60 kDa mitochondrial heat shock protein
stress-70 protein
60 kDa mitochondrial heat shock protein 9, 5
stress-70 protein
a This table represents the proteins identified from three different
concentrations of Az-HNE applied exogenously to intact RKO cells.
b The number value indicates the percent amino acid coverage of each
protein identified by peptide fragmentation using LC-MS/MS analysis.
a Percent coverage of select proteins from experiments using Al-HNE
adducted proteins from RKO cells (2 mg of protein in cell lysates).
b Percent coverage of proteins selected from Az-HNE adducted proteins
in RKO cell lysates.
dramatically over that of the control. This suggests that although
these highly abundant proteins are identified in controls, they
are specific for Al-HNE and Az-HNE modification. Further-
more, the percent coverage and overall similarity between the
proteins identified using both Al-HNE or Az-HNE confirms that
this method can reliably identify proteins adducted by HNE.
Discussion
This study reports the application of two different methods
for labeling HNE-adducted proteins with biotin for subsequent
enrichment and identification by proteomic analysis. The first
method attempted to utilize the modified Staudinger ligation
for the biotin conjugation of Az-HNE-adducted proteins. The
Staudinger ligation selectively couples an azide with triph-
enylphosphine by reaction of a methyl ester ortho to triph-
enylphosphine, which captures the nucleophilic aza-ylide by
intramolecular cyclization to form a stable amide bond and a
phosphine oxide (Scheme 1) (28). Using BiotTPhPh, we
specifically labeled Az-HNE-adducted proteins in lysates of
RKO cells very efficiently with biotin (Figure 3). However, the
results showed that biotin-conjugated proteins were not eluted
from SA beads until their denaturation by boiling (lane 9, Figure
4B), which also resulted in the elution of nonspecific proteins
(lane 9, Figure 4C and D). Attempts to remove the nonspecific
proteins with very stringent washes, such as 5 M NaCl, 5%
Igepal, or 4 M guanidine-HCl, failed (data not shown). It is
unknown why the biotin-conjugated proteins did not elute in
the 70% CH3CN and 5% FA fraction, which was relatively free
of nonlabeled proteins. The lack of affinity purification of these
biotin-tagged proteins directed us to using click chemistry as
an alternative to the modified Staudinger ligation for conjugating
biotin to HNE-adducted proteins.
Figure 7. Streptavidin purification of Al-HNE adducted proteins
conjugated with biotin. Cellular lysates (lane 1) containing Al-HNE
adducted proteins conjugated with biotin (2 mg) were incubated with
1 mL of SA beads (1:1 in ammonium bicarbonate) for 2 h. After
2 h, the beads were recovered by centrifugation, and the break-
through was collected (lane 2). Successive washes include 1% SDS
(lane 3), 4 M urea (lane 4), and 1 M NaCl (lane 5). Elution of biotin-
tagged proteins was done with 70% CH3CN and 5% formic acid
(lane 6). Biotin-tagged proteins were detected with HRP-SA, and
2.5 µg of protein was loaded per lane. Panel A shows the control
(DMSO) and 5 µM Al-HNE, and panel B shows 10 and 50 µM
Al-HNE. S, biotin-labeled standard curve.
Information (Tables S2 and S3). Table 3 shows that the same
stress signaling and redox regulatory proteins were found from
both Al- and Az-HNE treatment of RKO cells. We found that
highly abundant proteins were identified in the DMSO control
samples when 2 mg of cell lysate was used. Hsp90R-2 was
found in control samples for Az-HNE-treated samples only,
whereas GRP78, 60 kDa mitochondrial heat shock protein, and
the stress-70 protein were identified in the control samples for
Az-HNE- and Al-HNE-treated cells. Peroxiredoxin-1 was
identified in control samples for Al-HNE-treated cells only.
However, the percent coverage for these proteins increased

442 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Vila et al.
The copper-catalyzed Huisgen 1,3-dipolar cycloaddition reac-
tion couples an azide with a terminal alkyne (35). Both azides
and alkynes are stable in cells and in vivo, thus providing good
probesforspecificallylabelingHNEmodifiedproteins(28,31,32,43).
The reaction between the click chemistry reagents Az-HNE and
Al-HNE with Al-Biot and Az-Biot, respectively, proceeded in
the presence of TCEP in crude cell lysates, whereas in the
peptide model studies, the reaction did not occur. Typically,
the reactants and products from click chemistry reactions appear
insoluble in aqueous buffer, yet the reaction proceeds more
efficiently than in organic solvent (34, 44). A potential explana-
tion for this, proposed by Kolb et al., is that organic molecules
that are poorly soluble in aqueous solution have higher reaction
kinetics because of a greater free energy than when soluble in
organic solvents (34). We also observe insoluble particulates
during click chemistry reactions in cellular lysates, thus
potentially explaining the observed efficient biotin labeling in
cell lysates compared to that in model peptide studies. However,
the model studies clearly show that Asc is the better choice for
a reductant for efficient click chemistry. Also, studies in cellular
lysates demonstrate that Asc is a better reductant for click
chemistry than TCEP. Others have used Asc as the reductant
for click chemistry in model systems (45), but it has not been
reported for biological systems. Biotin-tagged proteins from
click chemistry reactions done in the presence of Asc were not
affinity purified with SA beads. As judged by Coomassie
staining, there was a similar profile of proteins that eluted from
SA beads incubated with the DMSO control and 50 µM Az-
HNE-treated samples. Metal-ion-catalyzed oxidation of proteins
from the presence of oxidants generated from CuSO4and Asc,
such as hydroxyl radical or superoxide (46), may have resulted
in enhanced binding with sepharose beads or caused the
oligomerization of proteins. Oligomerization of proteins to
biotin-tagged proteins would result in the appearance of
nonspecific proteins following affinity purification steps. There-
fore, TCEP was used for further experiments rather than
ascorbate as the reductant for click chemistry.
in vivo studies was the use of NaCNBH3 following cell lysis
to stabilize Az- and Al-HNE Schiff base adducts on Lys residues
of proteins (47). However, reversible Michael adducts on Lys
residues must be stabilized with NaBH4 (14). Therefore, some
Michael adducts to Lys may have been lost during workup
procedures prior to proteomic analysis. Further studies will be
aimed at characterizing HNE adducts on proteins using NaBH4
to ensure the recovery of all adducts. Nonetheless, the method
described in this study provides an unambiguous analysis of
proteins modified by HNE in vivo.
This study identified several proteins that were identified as
in vivo targets of activated derivatives of xenobiotics. Bro-
mobenzene and mycophenolic acid adduct to protein disulfide
isomerase isoform-A3 (PDIA3) in rat liver (48, 49). The small
molecule, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), was
shown to adduct the 60 kDa mitochondrial heat shock protein
and the stress-70 protein in rat kidney (50). Both halothane and
bromobenzene were shown to adduct GRP-78 in rat liver
(49, 51). Glutathione-S-transferase-Pi was adducted by acetami-
nophen and BHT in mouse liver (52, 53). Hsp-90R was adducted
by BHT in mouse lung (53), and peroxiredoxin-1 was adducted
by acetaminophen in mouse liver (52). HNE has been previously
shown to adduct to Hsp70, Hsp90, and PDIA3 (15–17), all of
which were also identified in this analysis.
A previous microarray experiment performed with RKO cells
treated with HNE, over the same concentration range used in
the present study, demonstrated that the expression of several
stress signaling proteins was induced via the heat shock response
and the ER stress response. Some of the proteins identified as
targets of HNE in the present study play important roles in the
control of these pathways. For example, several heat shock
proteins (i.e., HSP-70, HSP-90, 60 kDa heat shock protein,
mitochondrial precursor) and 78 kDa glucose-regulated protein
precursor (GRP-78) (a regulator or ER stress), and stress-70
protein (mitochondrial protein involved in protein binding and
cellular proliferation) were identified. GRP-78 and stress-70
protein were both strong hits with 8–11 unique peptides
identified in lysates from cells treated with 50 µM Az-HNE
(Table 1). Also, both proteins were identified at all Az-HNE
concentrations used (50, 10, and 5 µM; Table 1). When using
2 mg of cellular lysate protein, several of the heat shock proteins
and redox regulatory proteins were identified at concentrations
as low as 5 µM Az- or Al-HNE. This method can be used for
an unbiased analysis of HNE-modified proteins in cells and in
vivo for attempting to understand the roles that protein
modification plays in gene expression changes in response to
oxidative stress.
Proteomic analysis of affinity-purified biotin-conjugated
proteins showed that similar proteins were identified for Al-
and Az-HNE treated RKO cells (Table 3). This is in accordance
with other studies demonstrating that phenyl sulfonate ester
probes modified with either an azide (PSN3) or an alkyne (PS≡)
used for activity-based protein profiling of serine esterases
resulted in the identification of similar proteins (31). Also, the
PSN3 probe conjugation with the rhodamine-alkyne tag (Rh≡)
occurred more readily than the PS≡ conjugation to the
rhodamine-azide tag (RhN3), but the latter reaction had fewer
nonspecific interactions. Both reactions were reported to be
complete within 1 h (31). By comparison, we observed a slower
rate for click chemistry in RKO crude cell lysates with Az-
HNE concentrations of 10 µM and lower (∼3–6 h). After 90
min, both 25 and 50 µM Az-HNE-treated samples were
completely labeled, similar to the other study (31). In the present
study, Al-HNE resulted in fewer nonspecific bands as judged
by Western blot analysis in the control samples compared to
that in Az-HNE-treated samples (lane 1, Figure 7A vs lane 1,
Figure 6A). However, we observed more proteins in the Al-
HNE-treated cells for the control, 5, 10, and 50 µM than for
the Az-HNE-treated cells (50, 276, 456, and 538 vs 21, 171,
67, and 322). The percent coverage for the select proteins listed
in Table 3 is similar between Al-HNE and Az-HNE sample
sets. Both the Az- and Al-HNE probes are comparable in the
proteins they modify and are similar in reactivity with peptides
and toxicity in cells. One discrepancy between the in vitro and
Acknowledgment. This research was supported by funds
from the National Institutes of Health (P01-ES013125). We are
grateful to Simona Codreanu, Elizabeth Burnette, Hansen Wong,
and Matt Szapacs for helpful discussions of proteomic analysis,
and Andrew Felts for the synthesis of BiotTPhPh.
Supporting Information Available: Data comparing the
adducts formed for HNE, Az-, and Al-HNE to Angiotension
(Figure S1); data comparing the rates of adduct formation to
three different peptides with an equimolar ratio of HNE:Az-
HNE:Al-HNE (Figure S2); data comparing the ratio of adduct
formation for HNE, Az-, and Al-HNE in the presence of an
equimolar mixture of peptides (Figure S3); table showing all
of the proteins identified from Az-HNE-adducted proteins
affinity purified from cell lysates containing 150 µg of cellular
protein (Table S1); and table showing all of the proteins
identified in an experiment comparing Al- and Az-HNE-

HNE Protein Targets
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 443
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