Identification of tyrosine nitration in UCH-L1 and GAPDH

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Research Article

Identification of tyrosine nitration in UCH-L1and GAPDH

Protein tyrosine nitration is a post-translational modification commonly used as a

marker of cellular oxidative stress associated with numerous pathophysiological condi-

tions. We focused on ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1) and glycer-

aldehyde-3-phosphate (GAPDH) which are high-abundant brain proteins that have been

identified to be highly susceptible to oxidative modification. Both UCH-L1 and GAPDH

have been linked to the pathogenesis of Alzheimer’s and Parkinson’s disease, however;

specific nitration sites have not been elucidated. Identification of specific nitration sites

and quantitation of endogenous nitrated proteins are important in correlating this

modification to disease pathology. In this study, purified UCH-L1 and GAPDH were

nitrated in vitro with peroxynitrite and the presence of nitrated proteins was confirmed

by anti-3-nitrotyrosine Western blots. Data-dependent LC-MS/MS analysis identified

several distinct tyrosine nitration sites in UCH-L1 (Tyr-80) and GAPDH (Tyr-47, Tyr-92,

and Tyr-312). Subsequent validation with synthetic peptides was conducted for selected

nitropeptides. An LC-MS/MS method was developed for semi-quantitative determination

of the synthetic nitropeptides: KGQEVSPKVY*(UCH-L1) and mFQY*DSTHGKF

(GAPDH). The nitropeptides were detectable in the mid-attomole range and the peak

area response was linear over three orders of magnitude. Targeted analysis of endo-

genous UCH-L1 and GAPDH nitration was then conducted in an in vivo second-hand

smoke rat model to evaluate the utility of this approach.

Keywords:

Brain injury / Glyceraldehyde-3-phosphate / Nitration / Oxidative stressAQ1 Second-hand smoke / Ubiquitin carboxyl terminal hydrolase-L1

DOI 10.1002/elps.201100133

1 Introduction

Tyrosine nitration of proteins is an important post-transla-

tional modification (PTM) commonly used as a marker of

cellular oxidative stress associated with the pathogenesis of

several neurodegenerative diseases, such as Alzheimer’s

disease (AD) [1, 2], Parkinson’s disease and amyotrophic

lateral sclerosis [3]. Nitration of tyrosine residues to form

3-nitrotyrosine (3-NT) is a covalent modification mediated by

reactive nitrogen species (RNS) produced during nitrosative

stress. One of these reactive nitrogen species includes

peroxynitrite (ONOO–) which is formed by the reaction of

a superoxide radical anion (�O2–) with nitric oxide (�NO) [4].

The effect of nitration on protein function has been studied

extensively [5–9]. Tyrosine nitration can alter protein

structure and function due to a shift in pKa from 10.1 to

7.2 of the phenolic-OH which imparts a negative charge and

additional bulkiness to the tyrosine residue [10] as well as

affects the hydrolipophilic balance of the protein. The

electron withdrawing NO2 group decreases the electron

density of the phenolic group of a tyrosine residue which, if

present within the interacting region between an enzyme

and its substrate or between a receptor and its ligand, could

diminish interaction thereby, affecting the function of the

protein [11]. The effect of tyrosine nitration on protein

activity has been reviewed [12, 13]. Peroxynitrite-mediated

nitration can alter protein tertiary structure and lead to

degradation by the 20S proteasome [14, 15]. However, highly

damaged proteins cannot be degraded leading to insoluble

aggregate formation [14, 16] which has been linked to the

pathogenesis of several diseases [17]. Tyrosine nitration can

These are not the final page numbers

Joy D. Guingab-Cagmat1,3

Steven M. Stevens2

Mary V. Ratliff3

Zhiqun Zhang1

Mark S. Gold 3

John Anagli1

Kevin K. W. Wang1

Firas H. Kobeissy1,3

1Center of Innovative Research,Banyan Biomarkers, Inc.,Alachua, FL, USA

2Department of Cell Biology,Microbiology, and MolecularBiology, University of SouthFlorida, Tampa, FL, USA

3Center for Neuroproteomics andBioanalytical Lab at theMcKnight Brain Institute,Department of Psychiatry,University of Florida,Gainesville, FL, USA

Received September 21, 2010

Revised April 6, 2011

Accepted April 11, 2011

Abbreviations: GAPDH, glyceraldehyde-3-phosphate; IP,

immunoprecipitation; SHS, second-hand smoke; TBST, TBS

Tween-20; 3-NT, 3-nitrotyrosine; UCH-L1, ubiquitin carboxyl

terminal hydrolase-L1 Color Online: See the article online to view Figs. 4–6 in color.

Correspondence: Dr. Joy D. Guingab-Cagmat, Center of Innova-

tive Research, Banyan Biomarkers, Inc, 12085 Research Drive,

Alachua, FL 32615, USA

E-mail: jguingab@banyanbio.com

Fax:1 386-518-6813

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2011, 32, 1–14 1

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Journal: ELPS Manuscript Number 201100133 Color Figures XXX� � � � � � � � � � � � � �

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also affect tyrosine phosphorylation [9, 15, 18] and competi-

tion between nitration and phosphorylation has been shown

previously [19].

Despite the chemical reactivity of peroxynitrite, endo-

genous protein tyrosine nitration has demonstrated a

certain degree of selectivity within certain proteins and

specific Tyr residues. Factors that influence preferential

nitration have been reviewed previously [20–22]. Interest-

ingly, neither the abundance of a protein nor the number of

Tyr residues within the protein can predict whether a

protein is a target of nitration [20]. There is no specific or

preferred amino acid sequence that promotes protein

nitration; however, the secondary structure and local envir-

onment of the tyrosine residues seem to influence site and

protein-specific nitration [21, 23] as well as the proximity of

the nitrating agent to tyrosine residues [8, 24–27].

Many non-specific NT antibody-based methods and 2-D

SDS PAGE immunochemical approaches have focused on

the detection and quantitation of endogenous 3-NT provid-

ing an assessment of overall nitration of proteins. This

methodology has been applied in human plasma [28] and in

human brain tissue samples [29, 30]. However, these

methods assessed global nitration and failed to discriminate

between individual proteins and determine nitration levels

of specific proteins. Identification of precise Tyr site and

extent of nitration is important to elucidate the association

of the protein modification to the pathogenic mechanism of

disease. Different proteomic methods have been developed

to detect nitroproteins and identify specific tyrosine nitra-

tion sites including 1-D or 2-D SDS-PAGE, Western blot-

ting, and immunoprecipitation (IP) to resolve and

preferentially enrich nitroproteins followed by MS/MS [11,

31, 32]. In our study, specific tyrosine nitration sites were

identified in in vitro-nitrated model proteins using LC-MS/

MS. Ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1)

and glyceraldehyde-3-phosphate (GAPDH) are brain-abun-

dant proteins that have been identified to be highly

susceptible to oxidative modification. UCH-L1 is a major

neuronal protein that constitutes about 2% of the total brain

soluble protein [33, 34]. It is involved in the degradation of

unfolded or damaged proteins through the proteasome-

mediated degradation pathway [35, 36]. GAPDH is involved

in glycolysis and has been linked to cell apoptosis. GAPDH

has been shown to be very susceptible to peroxynitrite-

induced damage [37]. Inactivation of GAPDH can lead to

ATP depletion and damaged GAPDH can promote inso-

luble protein aggregation leading to cell death [38]. Both

UCH-L1 and GAPDH have been identified to be oxidatively

modified proteins in Alzheimer’s disease using redox

proteomics [1, 2, 36]; however, exact nitration sites of these

proteins have not been elucidated.

Second-hand smoke (SHS) exposure, one of the most

under-studied health risks, adversely affects a number of

vital human organs. While cardiac function abnormalities

and lung cancer due to SHS have been well characterized,

the effect of SHS on the brain has not undergone a full

systematic evaluation. It has been shown that smoking

induces peroxynitrite formation leading to oxidative stress

injury and nitration in vascular and lung tissue. In this

study, we propose to utilize SHS, as a proof of concept in

vivo model to translate our findings from the in vitro study

and to devise a MS-based quantification method that is

compatible with in vivo studies such as SHS.

2 Materials and methods

2.1 Identification of nitration sites in model proteins:

UCH-L1 and GAPDH

2.1.1 In vitro protein nitration

Purified rabbit muscle GAPDH was obtained from Sigma

(Sigma-Aldrich, MO, USA), whereas human recombinant

UCH-L1 was generated in-house. Peroxynitrite was obtained

from Upstate (Millipore, MA, USA). Solutions of 1 mg/mL

protein were prepared from the stock solutions by dilution

with 1� PBS. Approximately, 20 mg (0.81 nmol) of UCH-L1

and 20 mg (0.56 nmol) of GAPDH were prepared for in vitro

nitration. Prior to adding the peroxynitrite, 20 mL 1� PBS

was added to each tube and vortexed. Nitration was done by

bolus addition of concentrated peroxynitrite (140 mM).

UCH-L1 was treated with 0.0 mL (control), 0.2 mL (0.7 mM),

and 0.7 mL (2.45 mM), whereas GAPDH was treated with

0.0 mL (control), 0.2 mL (0.7 mM), 0.7 mL (2.45 mM), and

1.0 uL (3.5 mM) of peroxynitrite. Each tube was mixed using

a vortex and stored at ÿ801C prior to gel electrophoresis.

2.1.2 Western blotting validation

Control and peroxynitrite-treated UCH-L1 and GAPDH

were separated on a Novex (Invitrogen, Carlsbad, CA, USA)

10–20% Tris-glycine gel (1 mm, 10-well) and transferred to a

polyvinylidene difluoride (PDVF) membrane (Biorad, CA,

USA) by the semi-dry method in a transfer buffer (39 mM

glycine, 48 mM Tris, and 5% methanol) at 20 V for 2 h at

room temperature. Following blocking with 5% nonfat milk

in TBS Tween-20 (TBST), the membrane was incubated

overnight at 41C with the primary antibody. The membrane

was washed with TBST, and incubated with the secondary

antibody for 1 h at room temperature followed by TBST

washing and incubating with streptavidin-conjugated alka-

line phosphatase. The membrane was washed with TBST

and developed by nitroblue tetrazolium and 5-bromo-4-

chloro-3-indolyl phosphate. Monoclonal anti-3-NT (Upstate)

was used as primary antibody at a dilution of 1:1000.

Secondary biotinylated antibodies (Amersham Biosciences,

UK) and streptavidin-conjugated alkaline phosphatase were

used at a dilution of 1:1000.

2.1.3 1-D SDS PAGE

Laemmli buffer (2� ) was added to control and in vitro-

nitrated protein and approximately 7.5 mg (0.30 nmol)

Electrophoresis 2011, 32, 1–142 J. D. Guingab-Cagmat et al.

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UCH-L1 and 3 mg (0.084 nmol) GAPDH were loaded

and resolved on a 10–20% gradient Tris-glycine gel at

120 V for 2 h. True Blue (GE Healthcare, PA, USA)

molecular marker was used to indicate apparent gel relative

molecular mass. The bands were visualized with Coomassie

Blue staining.

2.1.4 Trypsin and chymotrypsin in-gel digestion

Gel bands were excised into 1 mm3 cubes, washed

thoroughly in HPLC-grade water (Burdick & Jackson,

Muskegon, MI, USA), washed with acetonitrile/water

(1:1 v/v), and dehydrated with acetonitrile. The gel cubes

were destained with acetonitrile/100 mM ammonium bicar-

bonate (1:1 v/v), and then dehydrated with acetonitrile. The

proteins were reduced with 100 mL of 45 mM dithiothreitol

(DTT) at 551C for 30 min. After cooling to room tempera-

ture, DTT was replaced with 100 mL of alkylating agent

(100 mM iodoacetamide) and incubated in the dark for

30 min. The gels were washed with 100 mL acetonitrile/

50 mM ammonium bicarbonate (1:1 v/v) and dehydrated

with acetonitrile. The gel cubes were rehydrated with 15 mL

of 12.5 ng/mL sequencing grade protease (trypsin or

chymotrypsin) (Promega, WI, USA) and kept on ice for

45 min. Twenty microliter of 50 mM ammonium bicarbo-

nate was added and digestion was performed at 371C

overnight. The resulting peptide-containing supernatant

was extracted and pooled with two 50% acetonitrile with

0.1% formic acid extractions. The tryptic peptide extracts

were centrifuged under vacuum until dryness and the

residue was reconstituted in 15 mL of water with 0.1% formic

2.1.5 Tyrosine nitration site identification by LC-MS/

Reversed-phase HPLC MS/MS was employed for the

detection and identification of specific Tyr nitration. Nano-

flow reversed-phase chromatography was performed on a

Nanoacquity Waters HPLC system (Waters, Milford, MA,

USA). Two microliter of each sample was loaded via the

autosampler onto a 5 mm Symmetry 180 mm� 20mm trap

column at 4 mL/min for 10 min. The sample plug was then

directed to a 1.7 mM BEH130 C18 100 mm� 100 mm

column at a flow rate of 250 nL/min. The mobile phase

consisted of solvent A (99% water/1% acetonitrile with 0.1%

formic acid) and solvent B (75% acetonitrile/25% water with

0.1% formic acid). Separation was achieved using a run time

of 111 min. The first linear gradient was from 2 to 40% B

over 90 min, the second linear gradient was from 40 to 80%

B over 5 min, and 80% B was held for 5 min before

returning to the initial mobile-phase composition (2% B).

Tandem mass spectra were collected on a Thermo LTQ-XL

(Thermo, San Jose, CA, USA) using a data-dependent

acquisition in which MS/MS spectra were acquired on

the five most abundant ions at a given chromatographic

time point.

2.1.6 Database search

MS/MS spectra were extracted by Xcalibur version 2.7.0.

Charge state deconvolution and deisotoping were not

performed. All MS/MS spectra were analyzed using

SEQUEST (Thermo; version SRF v. 5) and X! Tandem

(www.thegpm.org; version 2007.01.01.1). SEQUEST

(v. sp3.1.1) was set up to search the trypsin and

chymotrypsin-indexed ipi.HUMAN (v3.59, 62 772 entries)

database for UCH-L1 samples and NCBI rabbit-specific

taxonomy database (33 062 entries) for GAPDH. X! Tandem

was set up to search a subset of the ipi.HUMAN database for

UCH-L1 samples and a subset of the NCBI rabbit database

for GAPDH samples also assuming both trypsin and

chymotrypsin digestion. SEQUEST and X! Tandem were

searched with a fragment ion mass tolerance of 1.00 Da and

a precursor ion tolerance of 2.5 Da. Carbamidomethylation

of cysteine was specified in SEQUEST and X! Tandem as a

fixed modification. Oxidation of methionine and nitration

of tyrosine to NT were specified in SEQUEST and X!

Tandem as variable modifications. Scaffold (version Scaf-

fold_2_04_00, Proteome Software, Portland, OR, USA) was

used to validate MS/MS-based peptide and protein identi-

fications. Peptide identifications were accepted if they could

be established at 495.0% probability as specified by the

Peptide Prophet algorithm [39]. Protein identifications were

accepted if they could be established at 499.9% probability

as determined by Protein Prophet algorithm and contained

at least two identified peptides [40]. Scaffold calculated 0%

false-positive rate using these filtering criteria.

2.1.7 Synthetic peptide validation

All spectra of identified putative nitropeptides with 95%

peptide probability were manually inspected for validity of

the identification. One peptide from each model protein was

chosen as a signature peptide. Native and nitrated versions

of the peptides were synthesized using FMOC chemistry

(NeoBioSci, MA, USA). The purity of the synthetic peptides

was determined by HPLC-UV in a trifluoroacetic acid (TFA)

system. All conditions were kept the same as the conditions

used in the LC-MS/MS identification of these nitropeptides

in nitrated UCH-L1 and GAPDH except a steep LC gradient

was used (resulting in a shorter LC-MS/MS acquisition

time). Each synthetic nitropeptide MS/MS spectrum was

compared to the corresponding identified nitropeptide MS/

MS spectrum.

2.1.8 LC-MS/MS quantitative analysis

A sensitive LC-MS/MS method was developed for the semi-

quantitative analysis of the signature nitropeptides. Calibra-

tion curves in mobile phase (water with 0.1% formic acid)

were generated by plotting the peak area of the sum of three

prominent fragment ions against the amount of the peptide.

Serial dilution of the peptide standards was performed using

water with 0.1% formic acid. LC-MS/MS analysis was

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performed using the same LC-MS/MS system described

above. Separation was achieved using a fast linear gradient

run at 500 nL/min and total analysis time of 29 min. MS/

MS detection of the peptides was done using full-scan MS/

MS mode. Instrument control and data acquisition were

done using Xcalibur 2.0 software. The peak area of

reconstructed ion chromatograms derived from the sum of

the three prominent fragment peaks of each peptide was

used for quantitation. Manual peak integration was done for

each analyte in the Xcalibur software.

2.2 Identification of endogenous-nitrated UCH-L1

and GAPDH in SHS rat model

2.2.1 SHS animal model

All procedures complied with the guidelines of the

University of Florida Institutional Animal Care and Use

Committee. Twelve-wk-old male Sprague–Dawley rats

(Harlem, Indianapolis, IN, USA) were randomized and

divided into control and experimental groups (n5 16). The

control group was exposed to room air, whereas experi-

mental group was exposed to tobacco smoke for 5 h/day for

5 days/wk for a total of 6 wk. A smoking machine (model

TE-10, Teague Enterprises, Davis, CA, USA) that closely

simulated passive smoking was used with a chronic

paradigm designed to mimic long-term exposure in

humans. Tobacco smoke was generated through burning

filtered Kentucky 1R4F reference cigarettes (Tobacco

Research Institute, University of Kentucky, Lexington, KY,

USA), and using a standardized smoking procedure (35 cm3

puff volume, 1 puff per minute, 2 s per puff, and 8 puffs per

cigarette). Mainstream and sidestream smoke were trans-

ported to a mixing and diluting chamber. The smoking

machine was adjusted to produce a mixture of 15%

mainstream smoke and 85% sidestream smoke. After

6 wk, the rats were deeply anesthetized with isoflurane

and sacrificed to collect brain regions.

2.2.2 Sample enrichment for protein nitration

Rat brain cortices were homogenized in lysis buffer

containing 1% v/v PBS, 1 mM DTT, and 1� protease

inhibitor cocktail (Roche). The tissue lysates were then

centrifuged at 8000� g for 5 min at 41C to clear and remove

insoluble debris. Protein concentrations were determined by

DC protein assay (Biorad). Control and SHS samples were

pooled (n5 6) to yield 1 mg total protein. The samples were

pretreated with 100 mL Protein A agarose bead slurry (Pierce,

USA) for 4 h at room temperature with gentle rocking and

the supernatant was collected for subsequent IP. IP of

nitrated proteins was done using 60 mL of NT affinity

sorbent gel slurry (Cayman Chemical) at 41C overnight with

gentle rocking. The beads were collected by brief centrifuga-

tion in a microcentrifuge and washed three times with 1�

PBS. The beads were resuspended in 50 mL Laemmli buffer

and boiled for 5 min. The samples were briefly centrifuged

to pellet the beads and the supernatant were loaded on a

10–20% Tris-glycine SDS gel. Western blotting with anti-3-

NT was done to confirm the presence of nitrated proteins

after IP.

2.2.3 Targeted LC-MS/MS analysis of nitrated GAPDH

and UCH-L1

Bands were cut between 24 and 38 kDa for LC-MS/MS

analysis. Samples were subjected to in-gel digestion with

chymotrypsin. For this experiment, a different LC-MS

system with a dual pressure ion trap mass spectrometer

was employed. Reversed-phase HPLC MS/MS was

employed for the detection of nitrated UCH-L1 and

GAPDH. Nanoflow reversed-phase chromatography was

performed on a Proxeon EasynLC system (Proxeon, SE).

Two microliter of each sample was loaded via the

autosampler onto a trap column (EASY-Column 2 cm,

100 mm id, 5 mm, C18-A1 [SC001] at 3 mL/min for 5 min).

The sample plug was then directed to an analytical

column (EASY-Column 10 cm, 75 mm id, 3 m, C18-A2

[SC200]) at a flow rate of 300 nL/min. The mobile

phase consisted of solvent A (100% water with 0.1% formic

acid) and solvent B (100% acetonitrile with 0.1%

formic acid). Separation was achieved using a run time of

100 min. The first linear gradient was from 2 to 40% B over

90 min, the second linear gradient was from 40 to

80% B over 5 min, and 80% B was held for 5 min before

returning to the initial mobile-phase composition (2% B).

Tandem mass spectra were collected on a Thermo

LTQ-Velos (San Jose, CA) using a data-dependent

acquisition in which MS/MS spectra were acquired on the

ten most abundant ions at a given chromatographic time

point. MS/MS spectra were searched against a chymotryp-

sin-indexed IPI rat database. All protein database search

parameters were the same as those used in the in vitro

experiments.

2.3 Results

2.3.1 Detection of in vitro-nitrated proteins

It is important to demonstrate that endogenous-nitrated

proteins are readily detected in brain tissue after in vitro

treatment with peroxynitrite. As a preliminary investigation,

Western blotting probed with monoclonal anti-NT was

performed to detect nitrated proteins in a rat cortex

lysate and assess the efficiency of in vitro nitration.

Increasing signal intensity was observed with increasing

peroxynitrite concentration (200–3000 mM) (Supporting

Information Fig. S1). Stability of in vitro-nitrated

proteins was observed after incubation in different

reducing agents including b-mercaptoethanol, gluthathione,

and DTT as indicated by Western blot (Supporting

Information Fig. S2).

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2.3.2 SDS-PAGE and Western blots of model proteins

Two high-abundance brain proteins, UCH-L1 and GAPDH,

were used as model proteins. 1-D SDS PAGE and Western

blots of in vitro-nitrated UCH-L1 and GAPDH are shown in

Fig. 1. In vitro nitration was carried out by bolus addition of

concentrated peroxynitrite to 10 mg of purified UCH-L1

(0.4 nmol) and GAPDH (0.28 nmol). Concentrations of

peroxynitrite were 0.7 and 2.5 mM in nitrated UCH-L1

and 0.7, 2.5, and 3.5 mM in treated GAPDH. The decrease

in the intensity of the gel stain and increase in the

intensity of the anti-3-NT signal compared with the control

indicate the extent of nitration in each model protein. The

decrease in the Coomassie-stained gel band intensities of

either protein was consistently observed after peroxynitrite

treatment (Fig. 1). The apparent change in the band

intensity can be attributed to the increase in the protein

heterogeneity due to tyrosine nitration as a result of

a shift in hydrolipophilic balance of the protein. Another

factor that can lead to decrease in band intensity is

aggregation of oxidized protein induced by in vitro

treatment with peroxynitrite as indicated by the presence

of high-molecular-weight bands following nitration

(Supporting Information Fig. S3). In this study, Western

blotting was performed for the validation of the nitrated

UCH-L1 and GAPDH.

Figure 2 shows the immunoblots of the peroxynite-

treated UCH-L1 and GAPDH probed with their corre-

sponding protein antibody and anti 3-NT. UCH-L1 and

GAPDH were nitrated in vitro and spiked into rat brain

lysates and detected. In the presence of a complex back-

ground matrix, the lowest amounts of protein detected were

2.5 mg (0.1 nmol) and 0.5 mg (0.014 nmol) UCH-L1 and

GAPDH, respectively.

2.3.3 Identification and validation of in vitro-nitrated

proteins

Top-5 data-dependent LC-MS/MS acquisition was

performed for the identification of putative nitrated peptides

in UCH-L1 and GAPDH. Putative nitrated peptide

sequences obtained from the top-5 data-dependent LC-MS/

MS experiment are summarized in Table 1. Manual

validation of all observed nitropeptide spectra resulted in

the identification of a total of one and three nitration sites

for UCH-L1 and GAPDH, respectively (Fig. 3). Although

both proteins were subjected to trypsin and chymotrypsin

in-gel digestion, following manual inspection of all spectra

of all identified nitropeptides, only one tryptic peptide

spectrum was definitively assigned in GAPDH and none in

UCH-L1.

A representative nitrated peptide from each model

protein was selected for further validation using a synthetic

peptide of the same sequence. The non-nitrated versions of

the peptides were also synthesized. Comparisons of the MS/

MS spectra of the identified and synthetic peptides shown in

Figure 1. 1-D SDS PAGE and Western blots of in vitro-nitrated model proteins (A) UCH-L1 and (B) GAPDH. In vitro nitration was done by

bolus addition of concentrated peroxynitrite to 10 mg of purified UCH-L1 and GAPDH. Concentrations of peroxynitrite were 700 and

2450 mM in treated UCH-L1 and 700, 2450, and 3500 mM in treated GAPDH. Shown on the right-hand side are the quantitative

assessments of in vitro nitration for each model protein.

UNCORRECTED P

Figs. 4 and 5 further provide support for the definitive

identification of the nitropeptides. An LC-MS/MS method

was developed for the semi-quantitation of these peptides.

The LC and MS parameters were optimized using a mixture

of the nitrated and non-nitrated peptides. A shorter 30 min,

LC-MS/MS analysis was employed for quantification of the

peptides. The calibrators were prepared by serial dilution of

peptide solutions. The peak area obtained from recon-

structed ion chromatograms derived from the sum of the

three prominent product ions was manually integrated for

quantitation (Fig. 6A). The MS/MS spectra of peptides

KGQEVSPKVY, KGQEVSPKVY�, mFQYDSTHGKF, and

mFQY�DSTHGKF were collected to confirm the identity of

the peptides (Fig. 6B). The peak area response was linear

over three orders of magnitude as shown in Fig. 7. The

estimated detection limits (S/NZ3) for all peptides were in

the mid-attomole region.

2.3.4 In vivo nitration in SHS rat model

The possibility and limits of extending the procedure to

complex mixture were evaluated. Enrichment procedures

prior to LC tandem identification of these low-abundance

nitrated proteins have been employed to detect them with

high selectivity and sensitivity [22, 41]. An IP method using

anti-NT-conjugated agarose beads was utilized to enrich NT

prior to LC-MS/MS analysis. Method development and

validation involved IP of in vitro-nitrated UCH-L1 and

GAPDH and a more complex in vitro-nitrated brain tissue

lysate (Supporting Information Fig. S4). The enrichment of

nitrated proteins was validated by Western blotting with

anti-3-NT. The IP method was applied to control and SHS

samples and the endogenous nitrated proteins were probed

by anti-3-NT. To minimize any nonspecific binding of other

proteins to the antibody, samples were pretreated with

Figure 2. Immunoblots show-

ing the in vitro-nitrated UCH-

L1 and GAPDH probed with

the corresponding protein

antibody and anti 3-NT. In

vitro-nitrated proteins were

detected in 10 mg rat brain

lysate spiked with nitrated

proteins. The lowest amounts

of nitrated protein in the brain

lysate detected were 2.5 mg

UCH-L1 and 0.5 mg GAPDH.

Table 1. List of nitrated peptides identified from the LC-MS/MS analysis of in vitro-nitrated UCH-L1 and GAPDHa)

Nitrated protein Digestion enzyme Putative nitrated sequence Corresponding nitrated residue

UCH-L1(human) Chymotrypsin KGQEVSPKVYb) Tyr-80

GAPDH(rabbit) Trypsin LISWY�DNEFGYSNR Tyr-312b)

Chymotrypsin mFQY�DSTHGKF Tyr-47b)

Chymotrypsin MFQY�DSTHGKF Tyr-47

Chymotrypsin QY�DSTHGKF Tyr-47

Chymotrypsin qERDPANIKWGDAGAEYb) Tyr-92b)

a) The listed unambiguous peptide sequences had 495% probability as determined by the Peptide Prophet algorithm. Asterisks indicate

nitration (144.98), m indicates oxidation of methione (116), and all cysteine residues shown were modified via carbamidomethylation

(157.02).

b) Equivalent to Tyr-314, Tyr-49, and Tyr-94 in human GAPDH.

UNCORRECTED P

50% v/v protein A agarose beads. A dramatic increase in

3-NT signal in the Western blot of the IP compared with the

non-IP samples indicates isolation and enrichment of the

nitrated proteins (Fig. 8A). NT signal increase was observed

in Western blot of the immunoprecipitated SHS relative to

the control sample (Fig. 8A). The presence of nitrated UCH-

L1 and GAPDH was confirmed by Western blot with their

corresponding antibodies (Fig. 8B and C). Coomassie-

stained bands that aligned with UCH-L1 at 25 kDa and

GAPDH at 36 kDa GAPDH were excised from the gel for

LC-MS/MS identification of nitrated proteins and specific

nitration sites. The SEQUEST search identified the two

target proteins; however, no specific nitration sites were

identified.

A quantitative assessment of the nitration levels of

control versus SHS is shown in Fig. 9. Visible bands on the

Coomassie-stained gel between 24 and 38 kDa were cut for

LC-MS/MS analysis. The presence of endogenous UCH-L1

and GAPDH in the 3-NT immunoprecipitate samples was

confirmed from the data-dependent LC-MS/MS; however,

no nitration sites were identified from this analysis. This

result indicates that nitrated peptides derived from in vivo

samples are still below the limit of detection by data-

dependent acquisition after enrichment of nitrated proteins.

Additionally, even with the enhanced sensitivity of the

targeted MS method developed for the identification of

nitrated peptides from in vitro experiments, nitration is

either below the limit of detection for targeted analysis or

tyrosine nitration selectivity is different for SHS-induced

nitrosative stress in vivo compared with in vitro nitration by

peroxynitrite carried out in this study. Overall, these data

taken together (Figs. 8 and 9) demonstrate that nitrated

protein detection by MS for an in vivo study can be

accomplished; however, future in vivo studies will focus on

further optimization of the targeted MS method for site-

specific detection and quantification of protein nitration.

3 Discussion

3.1 In vitro tyrosine nitration

Previous studies have identified several endogenous

proteins to be highly susceptible to nitration under several

pathological conditions in animal models [1, 42–44]. To date,

information is limited on the identification of specific

nitration sites which can provide more useful insights about

the mechanisms of these diseases [2, 3, 5, 45, 46]. In this

study, specific in vitro nitration sites were identified in

model proteins, UCH-L1 and GAPDH. Both UCH-L1 and

GAPDH are high-abundance proteins that have been

identified to be highly susceptible to oxidative modification

and have been linked to disease pathogenesis; however,

specific nitration sites have not been elucidated. Using the

combination of SDS-PAGE, Western Blotting and LC-MS/

MS analysis, one and three unique nitration sites were

Figure 3. Amino acid sequence and a linear model of human UCH-L1 (accession P09936 ) (A) and rabbit GAPDH (accession P46406) (B)

showing the tyrosine nitration sites. Nitrated peptides (bold) and tyrosine sites (underlined) are highlighted. The linear models show the

identified tyrosine sites and the protein active sites.

UNCORRECTED P

57Figure

4.SidebysidecomparisonsoftheMS/M

SspectraoftheidentifiedUCH-L1-nitratedpeptideKGQEVSPKVY�(A

)versusthesyntheticpeptideKGQEVSPKVY�(B)andtheidentified

UCH-L1non-nitratedpeptideKGQEVSPKVY

(C)versusthesyntheticpeptideKGQEVSPKVY

UNCORRECTED P

57Figure

5.SidebysidecomparisonsoftheMSMS

spectraoftheidentifiedGAPDH-nitrated

peptidemFQY�DSTHGKF(A

)versusthesyntheticpeptidemFQY�DSTHGKF(B)andthe

identifiedGAPDH

non-nitratedpeptidemFQY�DSTHGKF(C)versusthesyntheticpeptidemFQY�DSTHGKF(D

UNCORRECTED P

57Figure

6.Extractedionchromatograms(A

)forthequantificationofUCH-L1peptidesKGQEVSPKVY�,KGQEVSPKVY,andGAPDHpeptidesmFQY�DSTHGKF,mFQYDSTHGKFgenerated

correspondingtandem

MSspectra.Chromatograms(A

generatedbyplottingthesignalofthreeproductions:m/z5

477,628,and854forKGQEVSPKVY�andKGQEVSPKVY,

596,999,and1071formFQY�DSTHGKF,andm/z5606,656,and954formFQYDSTHGKF.CollectedMS/M

Sspectraare

shown(B)to

confirm

theidentity

ofthepeptides.Data

acquiredusingamixture

ofthefourpeptidesat0.25ng/mL.A

seven-pointGaussiansmoothingwasapplied.Y�,nitratedtyrosineresidue.RT,retentiontime;MA,manuallyintegrated

area;andSN,signal-to-noise.

UNCORRECTED P

identified in in vitro-nitrated UCH-L1 and GAPDH,

respectively. The peptide mass tolerance was set to 2.5 Da

which is typically considered for database search of MS/MS

spectra acquired from an ion trap instrument such as an

LTQ XL. The database search resulted in a list of putative

nitrated peptides at 95% probability as determined by the

Figure 7. Linear regression analyses (n5 3) of UCH-L1 (A) and GAPDH (B) peptide semi-quantitation by LC-MS/MS. The ranges of

concentration were from 0.005 to 10 ng/mL for UCH-L1 and from 0.025 to 10 ng/mL for GAPDH. Chromatographic response remained

linear over three orders of magnitude for all four peptides. The estimated detection limit (S/NZ3) is in the mid-attomole region. Log–log

plots are generated to show the lower limits (inset).

Figure 8. IP enrichment of endogenous nitrated protein in SHS exposed rat cortex samples (pooled n5 6). Shown are targeted

immunoblotting analyses of nonenriched (No IP) and enriched (IP) samples. Ten microgram nonenriched samples and the collected IP

eluates from 500 mg samples were resolved on 1-D SDS-PAGE followed by Western blotting with (A) anti-3-NT, (B) anti-UCH-L1, and

(C) anti-GAPDH.

UNCORRECTED P

Peptide Prophet Algorithm and each spectrum was then

manually inspected. The in vitro-nitrated peptides listed in

Table 1 were subjected to manual validation. Assignment of

tyrosine nitration can be difficult when using low-mass

accuracy-based methods such as data-dependent scan on a

linear ion trap because of a combination of several factors

that can lead to isobaric interference. Manual validation of

the MS/MS spectra still plays an important role to

definitively assign these spectra to the correct peptide

sequences. Stevens et al. described the factors that can lead

to misidentification of tyrosine nitration by shotgun

proteomics and devised a general protocol for manual

validation of MS/MS-based identification of tyrosine nitra-

tion [47]. MS/MS spectra from putative nitrated peptides

were manually validated using theoretical CID MS/MS

fragment ions generated by the in silico peptide fragmenta-

tion program MS-Product (prospector.ucsf.edu). In our in

vitro experiment, the unmodified cognate peptide of each

nitrated peptide identified was readily detected. In this

study, both trypsin and chymotrypsin were used for

proteolysis to increase sequence coverage by MS analysis.

Trypsin was first used for in-gel digestion and resulted in

short tryptic nitropeptides. However, tryptic peptides shorter

than seven amino acids are not recommended for nitration

identification (and sequence identification in general) due to

the limited structural information for identification [47]. The

GAPDH tryptic peptide LISWY�DNEFGYSNR was the only

nitrated tryptic peptide reported in this study. As chymo-

trypsin digestion resulted in longer peptides, most of the

listed putative nitropeptides are chymotryptic peptides. In

this study, UCH-L1 and GAPDH were nitrated in vitro with

increasing concentration of peroxynitrite. The LC-MS/MS

experiment revealed no nitrated peptides in the control

samples (Supporting Information Figs. S5 and S6). The

reported putative nitrated peptides in Table 1 were all

identified in either the protein treated with the highest

concentration of peroxynitrite, or 2.5 mM ONOO– and

3.5 mM for UCH-L1 and GAPDH, respectively. All the

listed peptide sequences had 495% probability as deter-

mined by the Peptide Prophet algorithm. A number of

nitrated peptides were identified at peptide identification

probabilities o95% or identified at Z95% peptide identi-

fication probability but did not pass manual validation and

were not included in Table 1; however, they are shown in

Supporting Information Figs. S5 and S6. No identifiable

nitrated peptides were present in the samples treated with

lower concentrations of peroxynitrite except for GAPDH

treated with 2.5 mM ONOO–.

UCH-L1 is composed of two tyrosine residues out of a

total of 223 amino acids. Nitrated Tyr-80 has been identified

in the chymotryptic peptide KGQEVSPKVY80� and suppor-

ted by putative identification on tryptic peptides VY80�FMK

and GQEVSPKY80�FMK. Tyr-80 is located on the surface of

the protein completely exposed for nitration and cleavage by

both trypsin and chymotrypsin. Tyr-173 on the other hand is

partially exposed to the surface of the protein. GAPDH is

Figure 9. Targeted identification of nitrated UCH-L1 and GAPDH in SHS rat cortex samples. Pooled control and SHS samples (n5 6,

1.0 mg protein total) were immunoprecipitated prior to resolving on 1-D SDS gel and Western blotting with anti-3-NT (A). A quantitative

assessment of the nitration levels for UCH-L1 and GAPDH is shown (B). Shown are the bands in the Coomassie-stained gel (C) for LC-MS/

MS-nitrated protein identification.

UNCORRECTED P

composed of 8 tyrosine residues out of 333 amino acids.

Nitrated Tyr-47, Try-92, and Tyr-312 residues are in

peptide sequences with no assigned secondary structure

and are fairly exposed. The other five tyrosine residues are

either within an a helix or extended strand participating in

b ladder. Interestingly, the MS/MS spectrum used for the

identification of Tyr-312 was almost accepted as incon-

clusive since a couple of peaks above 20% relative intensity

were unidentified after the database search (Supporting

Information Fig. S7A). We found after careful inspection

that the MS/MS seems to be a distribution of the nitrated

tryptic peptides LISWY�DNEFGYSNR and LISWYDNEF-

GY�SNR that coeluted during LC-MS/MS analysis. Based

on the lower intensity of fragment ion peaks correspond-

ing to nitration at Tyr-318 (LISWYDNEFGY�SNR), we

hypothesize that nitration selectivity was greater for

Tyr-312 on GAPDH compared with the nearby tyrosine

residue at Tyr-318. Additionally, no doubly nitrated tryptic

peptide was identified which could be due to decreased

solubility or alteration in ionization efficiency upon

multiple nitration events.

UCH-L1 had 50% (one out of two), whereas GAPDH

had 38% (three out of eight) of tyrosine that is nitrated. Not

all GAPDH tyrosine residues in the unmodified form were

detected and hence the percentage of nitrated tyrosine

residues calculated may not reflect the actual nitration level

of the protein due to limited sequence coverage from the MS

analysis. None of the identified nitration sites is within the

catalytic region of the enzymes.

Peptide synthesis is an important step in nitration site

validation providing supporting evidence for the definitive

identification of the putative nitrated peptides. Signature

peptides were selected for each model protein. Synthetic

versions of the non-nitrated and nitrated UCH-L1 peptides

(KGQEVSPKVY and KGQEVSPKVY�) and GAPDH

peptides (mFQYDSTHGKF and mFQY�DSTHGKF) were

synthesized using FMOC chemistry. All these peptides were

analyzed by MS/MS and spectra were compared with the

spectra derived from the nitration sites identified from the

LC-MS/MS experiment. A sensitive LC-MS/MS method was

developed for targeted semi-quantitative analysis of these

nitrated proteins. It is important to first demonstrate that

the target peptides were readily detected at low concentra-

tions. Mixtures of the unmodified and nitrated peptides

were prepared in 0.1% formic acid to optimize the LC and

MS/MS parameters. The peptides were detectable as low as

at mid-attomole levels. The peak response remained linear

over three orders of magnitude.

3.2 In vivo tyrosine nitration

A targeted approach to identify in vivo-nitrated UCH-L1 and

GAPDH was performed through a combination of IP, 1-D

SDS-PAGE, Western blotting, and LC-MS/MS. Due to the

low abundance of in vivo-nitrated proteins; it is difficult to

identify the specific nitrated tyrosine sites without applica-

tion of a suitable and efficient enrichment method. Here,

NT immunoaffinity precipitation was utilized to preferen-

tially enrich for nitrated proteins and 1-D SDS fractionation

was used to further decrease the complexity of the

immunoprecipitates prior to LC-MS/MS. The target

proteins GAPDH and UCH-L1 were identified yet no

specific tyrosine nitration sites were detected.

4 Concluding remarks

In summary, the present study reports the identification and

validation of unique tyrosine nitration sites in UCH-L1 and

GAPDH (Figs. 1–3), two potential targets for oxidative

damage in neurodegeneration. We further developed target-

specific nitropeptide-based quantification for these two

proteins (Figs. 4–7). Finally, to evaluate the possibility and

limits of extending the method to complex mixtures,

identification of UCH-L1 and GAPDH nitration induced by

SHS was also conducted in rat brain tissue (Figs. 8 and 9).

Target endogenous nitrated proteins were identified after

isolating and enriching for nitrated proteins. Protein assay of

the IP fractions showed that 8–12% of the total protein was

recovered from IP. Interestingly, Western blot assay esti-

mated 20 pmol UCH-L1 and GAPDH in the IP fraction

which indicates that the amounts are within the range of

sensitivity of the peptide-based semi-quantitative method.

However, it is important to note that the peptide digests

contained a mixture of non-nitrated and low-abundance

nitrated peptides. In a recent study, a similar IP methodology

has been employed and the presence of endogenous nitrated

proteins was detected by Western blotting [48]. Nitrated

proteins were identified; however, no MS/MS spectrum

corresponding to a nitrated peptide was identified by shotgun

LC-MS/MS. For future studies, more efficient enrichment

steps have to be developed and optimized (e.g. nitropeptide

immunoaffinity enrichment after digestion of enriched

nitrated proteins) in order to simplify the complexity of the

sample, thereby increasing the probability of identification of

specific nitration sites. The current semi-quantitative method

developed established that the signature in vitro-nitrated

peptides was detectable at sub femtomole levels. However, for

targeted detection of in vivo nitration, future studies are

needed to further investigate nitration selectivity since this

will significantly affect the development of targeted MS

methods derived from in vitro nitration experiments.

Additionally, isotopically labeled nitrated peptides will be

synthesized as internal standards to allow for the quantitative

assessment of endogenous nitration in biological samples

and nitration changes that may occur at specific tyrosine

residues due to nitrosative stress.

This research work was performed with support of the UF

Center for Nano Biosensors (CNBS) and Flight Attendant

Medical Research Institute (FAMRI).

The authors have declared no conflict of interest.

UNCORRECTED P

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