Age-associated tyrosine nitration of rat skeletal muscle glycogen

phosphorylase b: characterization by HPLC–nanoelectrospray–Tandem

mass spectrometry

Victor S. Sharov a, Nadezhda A. Galeva b, Jaroslaw Kanski a,

Todd D. Williams b, Christian Schoneich a,*

a Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047, USAb Mass Spectrometry Laboratory, University of Kansas, Lawrence, KS 66047, USA

Received 9 December 2005; received in revised form 23 February 2006; accepted 28 February 2006

Abstract

We identified age-dependent post-translational modifications of skeletal muscle glycogen phosphorylase b (Ph-b), isolated from F1 hybrids of

Fisher 344!Brown Norway rats. Ph-b isolated from 34 months old rats showed a statistically significant decrease in specific activity compared to

6 months old animals: 13.8G0.7 vs. 20.6G0.8 UmgK1 protein, respectively. Western blot analysis of the purified Ph-b with anti-3-NT antibodies

revealed an age-dependent accumulation of 3-nitrotyrosine (3-NT), quantified by reverse-phase HPLC–UV analysis to increase from 0.05G0.03

to 0.34G0.11 (mol 3-NT/mol Ph-b) for 6 vs. 34 months old rats, respectively. HPLC–nanoelectrospray ionization—tandem mass spectrometry

revealed the accumulation of 3-NT on Tyr113, Tyr161 and Tyr573. While nitration of Tyr113 was detected for both young and old rats, 3-NT at

positions 161 and 573 was identified only for Ph-b isolated from 34 months old rats. The sequence of the rat muscle Ph-b was corrected based on

our protein sequence mapping and a custom rat PHS2 sequence containing 17 differently located amino acid residues was used instead of the

database sequence. The in vitro reaction of peroxynitrite with Ph-b resulted in the nitration of multiple Tyr residues at positions 51, 52, 113, 155,

185, 203, 262, 280, 404, 473, 731, and 732. Thus, the in vitro nitration conditions only mimic the nitration of a single Tyr residue observed in vivo

suggesting alternative pathways controlling the accumulation of 3-NT in vivo. Our data show a correlation of age-dependent 3-NT accumulation

with Ph-b inactivation.

q 2006 Elsevier Inc. All rights reserved.

Keywords: Aging; Rat skeletal muscle; Glycogen phosphorylase b; Oxidative posttranslational modifications; 3-Nitrotyrosine; Mass spectrometry

1. Introduction

The post-translational oxidative modification of proteins has

been implicated in the age-dependent loss of physiological

function (Starke-Reed and Oliver, 1989; Stadtman, 1992;

Sohal et al., 1993). The accumulation of modified proteins is

species-and tissue-specific, controlled by a variety of factors

such as nature of the oxidant, antioxidant protection, protein

repair and protein turnover (Ji et al., 1990; Lawler et al., 1993;

Larsen, 1993; Reiter, 1995; Parkes et al., 1998; Sun and Tower,

1999; Arking et al., 2000; Leeuwenburgh et al., 1994;

Vigouroux et al., 2004; Mary et al., 2004; Grune et al.,

2004). Although, elevated tissue levels of protein carbonyls

0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.exger.2006.02.012

* Corresponding author. Tel.: C1 785 864 4880; fax: C1 785 864 5736.

E-mail address: schoneic@ku.edu (C. Schoneich).

(Yan et al., 1997, Yan and Sohal 1998) and 3-nitrotyrosine (3-

NT) (van der Loo et al., 2000; Paik et al., 2001; Drew and

Leeuwenburgh, 2002) as a result of aging and age-associated

pathology have been reported, the physiological significance of

these modifications must be addressed via the detailed

characterization of individual modified proteins. In fact, only

a limited number of papers specifically analyzed the age-

dependent posttranslational oxidative modifications of purified

proteins and their functional significance. In a few studies, Cys

oxidation of phosphoglycerate kynase (Gafni, 1981) or

sarcoplasmic reticulum Ca-ATPase (SERCA) (Viner et al.

1997, 1999), Met sulfoxidation of calmodulin (Gao et al.,

1998), Tyr nitration of SERCA2a (Viner et al., 1996, 1999;

Knyushko et al., 2005), flotillin-1 and a-tubulin (Dremina

et al., 2005) and actin (Aslan et al., 2003), or carbonylation of

mitochondrial aconitase (Das et al., 2001), adenine nucleotide

translocase (Yan and Sohal, 1998) and carbonic anhydrase III

(Cabiscol and Levine, 1995) have been characterized. An

Experimental Gerontology 41 (2006) 407–416

www.elsevier.com/locate/expgero

V.S. Sharov et al. / Experimental Gerontology 41 (2006) 407–416408

important message from these studies is a remarkable

selectivity of the age-dependent oxidative protein modifi-

cation. More recently, larger scale proteomic analyses

identified a number of proteins sensitive to oxidative/nitrative

modifications in vivo as a result of biological aging

(Schoneich, 2003; Kanski et al., 2003, 2005a,b; Castegna

et al., 2002; Poggioli et al., 2004; Levine, 2002). In our

laboratory, we characterized the sequence-specific age-depen-

dent tyrosine nitration of several rat skeletal muscle proteins,

such as b-enolase, a-fructose aldolase, creatine kinase and

glycogen phosphorylase b (Ph-b) (Kanski et al., 2005b), which

perform important functions in energy metabolism, suggesting

that the nitration of such key proteins can be, in part,

responsible for the well-described age-associated decline of

muscle motor function (Larsson and Ansved, 1995). The

detection of such age-associated site-specific protein tyrosine

nitration on a proteomic scale serves as a first step before a

more complete characterization of these protein modifications

and their physiological consequences on individual purified

proteins. Therefore, in the current paper, we analyzed the age-

dependent accumulation of 3-NT, oxidative modification of

Cys residues, and the enzymatic activity of Ph-b isolated from

the skeletal muscle of 6 and 34 months old F1 hybrids of Fisher

344 and Brown Norway rats (F344!BN F1), an established

animal model of longevity (Fernandes et al., 1997). This

protein plays a key role in myophosporylase deficiency

(McArdle’s disease), a common glycogenosis affecting skeletal

muscle function. The latter is caused by a number of mutations

or splicing of the muscle glycogen phosphorylase gene

(PYGM), which affect protein activity (Vorgerd et al., 1998;

Di Mauro et al., 2002). Given that aging is generally

characterized by a progressive loss of skeletal muscle

performance, the accumulation of oxidative and/or nitrative

modifications on muscle Ph-b may represent a potential

molecular basis for this phenomenon.

2. Materials and methods

2.1. Chemicals

TPCK-treated sequence grade trypsin was from Promega

(Madison, WI). Rabbit muscle Ph-b, bovine serum albumin

(BSA), dithiothreitol (DTT), SDS, 5,5 0-dithio-bis-(2-nitroben-

zoic acid) (DTNB), urea, NADP, rabbit liver glycogen,

adenosine-5 0-monophosphate, glucose-6-phosphate dehydro-

genase and sodium iodoacetate were purchased from Sigma

(St. Louis, MO). Phosphoglucomutase was obtained from

Boeringer Mannheim (Indianapolis, IN). Pre-cast Novexw tris–

glycine-SDS gels, molecular weight standard Mark12, running

and sample buffers were from Invitrogen (Carlsbad, CA).

Monoclonal antibodies against protein-bound 3-NT (clone

1A6) were from Upstate Biotechnology (Lake Placid, NY). A

maleimide-based fluorescence dye, ThioGlo1, was obtained

from Covalent Associates (Woburn, MA). All other chemicals

of highest commercially available grade were obtained from

Fisher (Pittsburgh, PA).

2.2. Animals

The research protocol outlined in this manuscript was

approved by the University of Kansas Animal Care Facility.

Young (6 months) and old (34 months) Fisher 344!Brown

Norway F1 hybrid rats were purchased from the National

Institute of Aging colonies maintained at Harlan Sprague-

Dawley, Inc. (Indianapolis, IN, USA). The rats were allowed to

adapt for 2 weeks after arrival in a 12 h light/dark cycle and

were provided with water and food ad libitum. The animals

were sacrificed by decapitation, and the muscle tissue rapidly

removed, immediately frozen in liquid nitrogen, and stored at

K80 8C.

2.3. Purification of rat skeletal muscle phosphorylase b

Isolation of Ph-b was performed essentially as described

earlier (Fisher and Krebs, 1962) with minor modifications as

outlined below. Briefly, frozen muscle tissue (5 g) was

thawed in 2.5 volumes of ice-cold isolation buffer (IB)

consisting of 20 mM Tris–HCl (pH 7.4), 1 mM EDTA,

0.5 mM DTT and 0.1 mM freshly added PMSF, homogen-

ized for 2 min at maximal speed in an ice-cooled Waring

blender LB10 (Waring, Torrington, CT) followed by 3!15 s sonication with a probe sonicator (Sonic Dismembrator

500, Fisher Scientific, Pittsburg, PA) at 10% power. The

homogenate was centrifuged at 8000g for 10 min; the

supernatant was saved and pooled with the supernatant of a

second extraction from the pellet with the same volume of

IB. Protein extracts were filtered through four layers of

cheesecloth and kept for 30 min at 37 8C for converting

phosphorylase a (Ph-a) to Ph-b and denaturing some

thermolabile proteins. Solid (NH4)2SO4 was slowly added

to precipitate proteins at 4 8C under stirring; the fractions

between 25 and 50% saturation after 6 h incubation were

collected by centrifugation at 20,000g for 20 min. Pellets

were dissolved in a small volume of IB and exhaustively

dialyzed against IB at 4 8C to remove the excess of

ammonium sulfate. The dialyzate was centrifuged at

20,000!g for 1 h to remove insoluble proteins, and applied

to a self-packed DEAE-Sephacel column (4.6!100 mm),

equilibrated with IB (in the cold cabinet). The column was

washed with this buffer until complete removal of unbound

proteins, and eluted by a linear gradient of NaCl from 0 to

0.3 M in ca. 10 bed volumes of IB at a flow rate of 1 ml/

min using an Acta Prime FPLC system (Amersham

Biosciences, Piscataway, NJ), equipped with absorbance

(monitoring at 280 nm) and conductivity detectors. Fractions

containing protein and Ph-b activity were pooled and further

separated/analyzed by SDS-PAGE and reverse-phase HPLC.

Protein concentration was determined by the bicinchoninic

acid (BCA) assay using BSA as a standard according to the

manufacturer’s instructions (Pierce, Rockford, IL). In some

experiments, where indicated, Ph-b isolation media did not

contain any reducing agents in order to maintain the

oxidation state of the protein isolated from muscle tissue.

V.S. Sharov et al. / Experimental Gerontology 41 (2006) 407–416 409

2.4. Gel electrophoresis

Samples of Ph-b containing 10–100 mg protein were mixed

with an equal volume of tris–glycine-SDS sample buffer

(containing 5% b-mercaptoethanol for reducing conditions),

heated for 2 min at 95 8C, and loaded into 1.5 mm thick 10-

well Novex 4–12% Tris–glycine gradient gels. After running

the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at

200 V for 90 min, the gels were stained with 0.2% Coomassie

R250 in 10% acetic acid/30% methanol/60% H2O (v/v/v) for

2 h followed by destaining in 7.5% acetic acid/40%

methanol/52.5% H2O (v/v/v) until the bands were visible

and the background was clear.

2.5. Reverse-phase HPLC of proteins

For reverse-phase HPLC separation of Ph-b, we used a

procedure described by us earlier (Sharov et al., 2002). Protein

samples were dissolved in neat TFA and injected onto Vydac

C4 column (250!4.6 mm id.), protected by a guard column

(10!4.6 mm id.), equilibrated with 40% (v/v) ethanol in 0.1%

aqueous TFA, and eluted at a flow rate of 0.5 ml/min by a

linear gradient increasing the ethanol content by 1.3%/min.

Chromatograms were monitored by absorbance in the spectral

range of 200–600 nm using a photodiode array (PDA) detector

(SPD-M10A) or by fluorescence using a fluorescence detector,

RF-10A (both from Shimadzu Corp., Japan).

The 3-NT content in Ph-b was estimated using the following

equation (Sharov et al., 2002)

3 � NT=Ph � bðmol=molÞZA360=A280!3280=3360;

where A280 and A360 are the Ph-b chromatographic peak areas

quantified at 280 (for protein) and 360 nm (for 3-NT), 3280Z116,720 MK1 cmK1 and 3360Z2100 MK1 cmK1 are the

respective molar extinction coefficients for Ph-b (calculated

from the protein sequence using the GPMAW 3.0 software

from ChemSW Inc., Fairfield, CA), and 3-NT at acidic pH

(Sharov et al., 2002). Similarly, the amount of ThioGlo1 (TG1)

attached to Ph-b Cys residues was quantified using the Ph-b

chromatographic peak areas detected at 379 and 280 nm

according to the following equation

TG1 � Cys=Ph � bðmol=molÞZA379=A280!3280=3379;

where A379 and 3379 are the Ph-b peak area and the extinction

coefficient (3379Z14,454 MK1 cmK1 according to the sup-

plier) for TG1-Cys adducts, respectively; A280 is the Ph-b

chromatographic peak area detected at 280 nm and 3280Z116,720 MK1 cmK1 (see above). The absolute amount of rat

skeletal muscle Ph-b was determined by both gel band

densitometry and RP-HPLC peak area using commercial

rabbit skeletal muscle Ph-b (Sigma, St Louis, MO) as an

authentic standard.

2.6. Assay of phosphorylase b activity

Ph-b activity was assayed at 21 8C in the direction

of glycogen degradation by coupling the production of

glucose-1-phosphate to NADP reduction by phosphoglucomu-

tase and glucose-6-phosphate dehydrogenase, essentially as

described earlier (Bergmeyer and Gawehn, 1974). The assay

medium contained 0.1 mM EDTA, 3 mM MgCl2, 0.3 mg mlK1

NADP, 0.2 mg mlK1 glycogen, 30 mM adenosine-5 0-mono-

phosphate, 0.7 U mlK1 phosphoglucomutase, and 3 U mlK1

glucose-6-phosphate dehydrogenase in 0.05 M potassium

phosphate buffer (pH 6.8). After recording a blank rate for

2 min, the reaction was initiated by the addition of Ph-b

samples, and the linear increase in absorbance at 340 nm was

monitored for 8 min. The activity was determined as DA340/

min and calibrated using the commercially rabbit skeletal

muscle Ph-b of known activity. To calculate the specific

activity of Ph-b in samples from young and old rats, data were

normalized for the protein quantity estimated by RP-HPLC of

respective samples, as described above.

2.7. Western Blot (WB) analysis

Proteins were transferred from gels to PVDF membranes by

electroblotting (at 100 V for 2 h). Primary antibodies

(mouse anti-3NT clone 1A6) were obtained from Upstate

Biotechnology (Lake Placid, NY). Peroxidase conjugated anti-

mouse IgG was purchased from Pierce (Rockford, IL), and the

blots were visualized with the ECL-plus WB detection kit from

Amersham Pharmacia Biotech (Piskataway, NY) as described

by the manufacturer. To assess specificity of protein 3-NT

detection with antibodies, two important controls were

employed, treatment with dithionite to reduce 3-NT on the

membrane prior to WB analysis, and blocking of the antibodies

with free 3-NT, as described elsewhere (Ye et al., 1996). Both

controls showed a negligible non-specific affinity of the 3-NT

antibodies to Ph-b bands.

2.8. In-gel digestion of Ph-b

Protein bands with apparent mass of 96 kDa were excised

from the gel, cut into pieces of about 1 mm3 size, and processed

as described elsewhere (Sharov et al., 2002). In brief, gel slices

were washed twice for 45 min at 37 8C in 0.5 ml of 200 mM

NH4HCO3/50% (v/v) acetonitrile with agitation. For protein

alkylation, the gel slices were incubated with 2 mM DTT in

200 mM NH4HCO3 for 30 min at 50 8C, followed by the

reaction with 5 mM iodoacetic acid for 30 min at room

temperature. After removal of the solvent the gel slices were

additionally washed for 1 h in 0.5 ml of 200 mM NH4HCO3/

50% acetonitrile (v/v) with agitation, and shrunk in pure

acetonitrile for 15 min. After removal of acetonitrile and

drying under vacuum, the samples were re-swollen with a

buffer containing 40 mM NH4HCO3, 1 mM CaCl2, 10% (v/v)

acetonitrile, and trypsin at a ca. 10:1 molar ratio of the protein

to trypsin (usually, 2 mg trypsin per gel band containing 20 mg

protein). The volume of buffer was ca. 1.5-times that of the

excised gel band. After the adsorption of trypsin, additional

buffer (30–50 mL) was used to cover the gel pieces during

overnight digestion (16–18 h) at 37 8C. For the extraction of

peptides from a gel, the overlay of the in-gel digest was

V.S. Sharov et al. / Experimental Gerontology 41 (2006) 407–416410

obtained after sonication for 30 min in a water bath of an

Ultrasonic Cleaner ME 4.6 (Mettler Electronics Corp.,

Anaheim, CA, USA) and short centrifugation, and used for

MS analysis.

2.9. Mass spectrometry

In-gel digests of Ph-b were analyzed by nano-HPLC with

electrospray ionization-tandem mass spectrometry (NESI-

MS/MS) using either a ThermoElectron LCQ Duo or a

ThermoElectron Classic (San Jose, CA) mass spectrometer

equipped with a nanoelectrospray source (Thermo Electron).

Separation of tryptic peptides was achieved on-line prior to

MS/MS analysis on in-house packed BioBasic C18 stationary

phase (Thermo Electron) nanoflow columns (300 A, 10 cm!75 mm, 15 mm tip size) (New Objective, Woburn, MA) with the

following chromatographic conditions: mobile phase A: 0.1%

formic acid in water, mobile phase B: 0.1% formic acid in

MeCN. The flow rate was 0.5 mL minK1, delivered by a

MicroTech Scientific Ultra Plus II pump (after 1:20 split), or by

a MicroTech Xtreme Simple nano-flow pump (direct flow).

The following gradient profile was used to increase mobile

phase B linearly to the following fractions: from 0 to 5 min

gradient held at 10% B, then increased to 60% B within

105 min, and continued at 60% B for additional 5 min. After

each run, the column was washed by a short gradient (0–60% B

for 20 min) and allowed to re-equilibrate to the initial

conditions for 15 min. The following instrumental conditions

were used for mass spectrometric analysis: number of

microscansZ3, length of microscansZ200 ms, capillary

temperatureZ160 8C, spray voltageZ1.9 kV, capillary volta-

geZ35 V, tube lens offsetZK14 V. The mass spectrometer

was tuned using the static nanospray setup with a 5 mM

solution of angiotensin I (MW 1296.5) infused by a pico-tip

emitter (New Objective). Data acquisition was performed in

the data-dependent fashion, i.e. an MS scan followed by 3 or 4

MS/MS scans of the 3 or 4 most intense peaks with the

normalized collision energy for MS/MS set at 35% and the

isolation width of 2.0 mzK1. A minimal signal for MS/MS

acquisition was set to 2!106. Additionally, the dynamic

exclusion option was enabled and set with the following

parameters: repeat countZ3, repeat duration 5 min, exclusion

list sizeZ25, exclusion durationZ5, and exclusion mass

widthZ3.

MS/MS data were analyzed using the Sequest algorithm

(Ducret et al., 1998) searching the most current non-redundant

NCBI protein database downloaded from the ftp.ncbi.nlm.nift-

gov/blast/db. The filter used for peptide identification included

correlation factors (XCorr) being greater than 1.5, 2.0, and 2.5

for the charge states C1,C2 and C3, respectively. In order to

identify post-translational modifications of rat Ph-b, database

search was performed against a custom database created of

only rat (or later mouse) Ph-b sequences. The following

modifications were accounted for during the search: oxidation

of Met (C16 amu; amuZatomic mass units), carboxymethyla-

tion (C58 amu) or oxidation of Cys (C32 and C48 amu), and

nitration of Tyr (C45 amu). Additionally, MS/MS spectra of

interest were examined manually for the presence 3-NT-

containing peptides (Kanski et al., 2005b). Analysis of the MS/

MS spectra was based on a search for the major sequence-

indicating ions resulting from the cleavage of the parent ion at

specific locations relative to the peptide bond. The Roepstorf-

Fohlman nomenclature (Roepstoff and Fohlman, 1984) was

used for the annotations of N-terminal (b) and C-terminal (y 00)

fragments.

2.10. Mapping rat Ph-b sequence using Q-TOF-MS/MS data

Rat Ph-b samples were subjected to in detail sequence

mapping using Waters CapLC XE system, Q-TOF mass

spectrometer 2 (Micromass, UK) and Protein Lynx Global

Server (PLGS 2.0.5) (Waters, Milford, MA). HPLC separation

of peptides derived from digestion of rat Ph-b with trypsin was

performed on RP-HPLC column (0.32!150 mm Symmetry

C18) at a flow rate of 8 ml minK1 with a linear gradient raising

from 25 to 75% (v/v) methanol in 0.08% (v/v) aqueous formic

acid over a period of 50 min. Ions of intensity greater than 4

counts in a survey scan were selected as precursors for MS/MS.

The obtained data were challenged against SWISS-PROT

databank using PLGS 2.0.5. The MS/MS data files were

processed using slow deisotopic deconvoluting parameters.

The initial PLGS workflow was amended by adding AutoMod

which allows non-specific cleavages and amino acid

substitutions.

MS/MS data obtained on both Q-TOF and LCQ instruments

were searched against a protein database using PLGS and

Sequest, respectively. Mouse skeletal muscle Ph-b

(PHS2_MOUSE, accession code Q9WUB3) was shown to be

the top hit returned by a simple databank search with a

coverage of up to 68%, while for rat skeletal muscle Ph-b

(PHS2_RAT, Swiss-Prot protein database accession code

P09812) only a 58% sequence coverage was obtained. To

resolve this discrepancy and improve mapping coverage,

multiple Ph-b in-gel digests were analyzed using CapLC and

Q-TOF instrumentation.

The databank search by PLGS with the enabled AutoMod

feature allowed us searching without enzymatic specificity and

to consider amino acid substitutions. The results demonstrated

that the experimentally obtained sequence of purified rat

skeletal muscle Ph-b differs from that published in the protein

database: we found 17 amino acid residue deviations at

sequence coverage of 67.8% (Fig. 1). These 17 experimentally

determined amino acid deviations from the published rat

sequence actually match the amino acids in the published

mouse Ph-b sequence. CLUSTAL W (1.82) multiple sequence

alignment (website http://www.ch.embnet.org/cgi-bin/clus-

talw_parser) of our experimentally obtained partial sequence

(496 out of 841 amino acids) with other database PHS2

sequences revealed that there were 492 identities for mouse,

485 identities for human and rabbit, 475 for bovine and sheep

sequences comparative to 479 identities for the rat sequence.

Because the experiment for rat Ph-b demonstrated a better

overlap with the published mouse than the rat sequence, the

mouse sequence was used in subsequent studies.

Fig. 1. Alignment of rat muscle Ph-b (PHS2_RAT) sequence from the Swiss-Prot database and sequence fragments obtained in our experiments. Highlighted italic

letters show differences in the rat Ph-b sequence relative to experimental MS/MS data. The dotted lines indicate peptides, which were not detected by MS/MS.

V.S. Sharov et al. / Experimental Gerontology 41 (2006) 407–416 411

2.11. Determination of DTNB-reactive Cys residues in Ph-b

For the quantitative analysis of reduced Cys residues in

Ph-b, the reaction with dithio-bis-dinitrobenzoic acid (DTNB)

was employed according to a well-established method

(Ellman, 1959) with minor modifications. Briefly, 100 mg of

dialyzed protein in 20 mM tris–HCl (pH 8.5) containing 1%

SDS and 0.2 mM DTNB was incubated for 30 min at 37 8C

and the absorbance at 412 nm was measured. A standard

curve was obtained using known concentrations of gluta-

thione (GSH) under the same conditions.

2.12. In vitro nitration of Ph-b

Purified Ph-b (1 mg mlK1 protein in 30 mM NH4HCO3, pH

7.8) was subjected to peroxynitrite at desired final concen-

tration up to 1 mM, as described earlier (Sharov et al., 2002;

Kanski et al., 2005b). The protein was then immediately

separated by SDS-PAGE and submitted to in-gel trypsin

digestion prior to NESI-MS/MS analysis, as described above.

2.13. Statistical analysis

Quantitative results were obtained from the data of at least

three independent experiments involving material isolated

from individual animals (nZ4 for each age group).

Values are presented as meanGstandard deviation. Signifi-

cance of a difference between two averages was assessed by

probability (P) associated with a Student’s t-test, calculated

using a two-sample unequal variance and two-tailed dis-

tribution with Microsoft Excel XP Pro software.

3. Results

3.1. Biochemical characterization of Ph-b Isolated from young

and old rat skeletal muscle

The purity of isolated Ph-b was assessed by SDS-PAGE

showing that anion-exchange chromatography yields pre-

parations significantly enriched with Ph-b (Fig. 2A). No

significant differences in apparent protein molecular weight

were observed between young (6 months old) and old (34

months old) samples. These preparations were used for the

analysis of enzyme activity. Further fractionation of the

enriched Ph-b samples by RP-HPLC (chromatograms are not

presented) and collection of the fractions eluted at 23–26 min.

yielded even more homogenous Ph-b preparations (Fig. 2B).

For quantitation of both Ph-b and 3-NT/Ph-b molar ratios, we

analyzed the RP-HPLC peak eluting at ca. 24.5 min., which

contains predominantly Ph-b (Fig. 2B). Analysis of 3-NT

molar content was done as described in the section 2.5. Based

on the independent isolation of Ph-b from 5 young and 5 old

animals, this analysis yields a statistically significant (P!0.05)

age-dependent accumulation of 3-NT on rat skeletal muscle

Ph-b. The 3-NT content rises from 0.05G0.03 mol 3-NT/mol

Ph-b in young to 0.34G0.11 mol 3-NT/mol Ph-b in old rats,

i.e. showing a ca. seven-fold increase (Fig. 3A). At the same

time, biological aging resulted in a ca. 33% decrease in the

Fig. 2. Characterization of the purified Ph-b from skeletal muscle of 6 (Y) and

34 (O) months old rats. (A) and (B): SDS-PAGE separation of Ph-b samples

after ion-exchange chromatography and after additional RP-HPLC purification,

respectively. (C) WB analysis of Ph-b immunoreactivity with anti-3-NT

antibodies. (D) Densitometry analysis of the immunoblots from panel C.

Protein load w 20 mg/lane in (A) and (B) or 4 mg/lane in (C). Data are

representative from at least three independent Ph-b purifications involving

muscle tissue from different rats.

V.S. Sharov et al. / Experimental Gerontology 41 (2006) 407–416412

specific activity of Ph-b (Fig. 3B) isolated from 34 months old

rats (13.8G0.7 U/mg protein) compared to 6 months old rats

(20.6G0.8 U/mg protein).

3.2. WB analysis of 3-NT content of Ph-b

Equal amounts of the purified protein from young and old

animals were resolved by SDS-PAGE. WB analysis demon-

strated a ca. 40% increase in 3-NT immunoreactivity for the

Ph-b bands obtained from 34 vs. 6 months old animals (Fig. 2C

and D). Reduction of the blots with sodium dithionite prior to

WB analysis abolished the 3-NT immunoreactivity (data not

shown), demonstrating the specificity of the anti-3-NT

antibody. The results of 3-NT quantitation on Ph-b by WB

analysis (Fig. 2D) are different from the data obtained by RP-

HPLC (Fig. 3A) largely due to higher background 3-NT

immunoreactivity in samples from young animals.

Fig. 3. Age-associated changes in Ph-b specific activity (A) and 3-NTcontent of

Ph-b assayed by HPLC–UV (B).

This apparent discrepancy may also be attributed to the semi-

quantitative nature of WB analysis; nevertheless, the WB

analysis qualitatively confirms the age-dependent increase in

the accumulation of 3-NT on rat skeletal muscle Ph-b.

3.3. MS analysis of Ph-b in-gel digests: identification of

sequences nitrated in vivo

Unambiguous proof for the 3-NT accumulation on Ph-b was

demonstrated by using NESI-MS/MS analysis. In three

independent experiments involving Ph-b isolated from three

different young and three different old rats, we identified 3

nitropeptides containing 3-NT at positions 113, 161 and 573.

MS/MS spectra of these nitropeptides are shown in Fig. 4A–C.

Peptides showing nitration of Tyr161, IHEY(NO2)KRQ

LLNC(O3)LHIITLYNR and Tyr573, Y(NO2)EFGIFNQ

KIC(O3)GGWQMEEADDWLR, were detected only in Ph-b

digests from old rats, whereas sequences containing nitrated

Tyr113, TLQNTMVNLALENACDEATY(NO2)QLDMEEL

EEIEEDAGLGNGGLGR, were identified in Ph-b from both

young and old animals at sufficiently high scores. For the latter

nitropeptide, multiple isoforms containing oxidized Met and/or

Cys residues were also observed although no age-dependent

tendencies in the accumulation of these oxidized peptides were

detected. For comparison and the generation of authentic

standards, we performed the reaction of purified rat Ph-b with

peroxynitrite in vitro.

3.4. Comparison of in vivo with in vitro nitration

Incubation of Ph-b isolated from 6 months old rat with

1 mM peroxynitrite resulted in an average formation of ca.

4.5 mol 3-NT/mol Ph-b, as monitored by RP-HPLC coupled to

UV detection (chromatograms not presented). At the same

time, multiple 3-NT-conaining peptides were detected from in-

gel tryptic digests. We found that the Xcorr values for

matching MS/MS data are useful to determine nitration site

selectivity. The ‘XCorr’ value represents a score calculated by

the Sequest algorithm, which cross-correlates experimental

MS/MS spectra from peptides with the theoretical mass spectra

produced from sequences generated from a protein database;

the higher the XCorr value, the higher the probability that a

given MS/MS spectrum belongs to a candidate peptide. Fig. 5

displays the XCorr values obtained for both Tyr and 3-NT-

containing Ph-b peptides showing protein tyrosine nitration at

multiple sites. For a qualitative analysis of the differential

sensitivity of individual Tyr residues towards nitration we used

only MS/MS spectra of XCorr values O1.5, O2.0, and O2.5

for Tyr- and 3-NT-containing peptides of the charge states

C1,C2 and C3, respectively. To compare the in vitro

nitration levels for individual Tyr residues, we classified

them into 3 groups: (1) significant levels of nitration, with

significant scores for the 3-NT-containing peptides (Tyr at

positions 51, 52, 113, 185, 203, 262, 280, 404, 473, 731, and

732), (2) insignificant nitration levels, where XCorr values for

the 3-NT-containing peptides are insignificant whereas the

XCorr values for respective native Tyr-containing peptides are

Fig. 4. 3-NT-containing sequences, detected by NESI-MS/MS, on Ph-b isolated

from 34 months old rats. (A)–(C) Representative tandem MS spectra for 3 Tyr

nitration sites in the sequence of rat skeletal muscle Ph-b. Inserts show the

respective sequences and decoding of the spectra.

V.S. Sharov et al. / Experimental Gerontology 41 (2006) 407–416 413

reliable (Tyr at positions 83, 84, 90, 155, 157, 161, 297, 511,

524, 548, 648, 726, 777, and 780), and (3) nitration levels that

cannot be resolved by our method, where the XCorr values for

both native and nitrated peptides were insignificant (Tyr at

positions 74, 75, 233, 374, 553, 573, 587, 613, 791, and 820).

Interestingly, all three of the 3-NT-containing peptides

detected in vivo did not fit to any single group classified

above. The Ph-b peptide containing Tyr113 can be significantly

nitrated in vitro. Peptides containing Tyr161 and Tyr537 were

not found in the nitrated form in vitro and not resolved at a

reliable score, respectively, although nitration in vitro yielded a

ca. 10 times higher levels of total 3-NT on Ph-b than those

observed in vivo (4.5 vs. 0.3 mol 3-NT per mol protein,

respectively). Most of the in vitro nitration-sensitive peptides

from group 1, except Tyr113-containing peptide, displayed no

significant accumulation of 3-NT in vivo. These data show that

the reactivity and access of peroxynitrite do not explain the

nitration of a given protein in vivo.

3.5. Analysis of reduced Cys residues in Ph-b

Determination of DTNB-reactive Cys residues did not

show a statistically significant age-dependent difference

between Ph-b purified from 6 and 34 months old rats (55.3G1.7 and 57.6G2.5 nmol SH/mg total protein, respectively).

Taking into account the Ph-b content in the fractions collected

after ion-exchange chromatography (ca. 60% based on gel

densitometry; Fig. 2A), we conclude that the Cys residues

in the purified Ph-b (ca. 8.9G0.3 and 9.3G0.4 mol Cys/mol

Ph-b, respectively) are virtually all in the reduced state. Our

values are close to the mouse muscle Ph-b content of 8 Cys per

protein molecule. The slight deviation of the experimental

values from the theoretical Cys number may be the result of

uncertainties in the gel densitometry and small level of

contaminating proteins. The HPLC analysis of ThioGlo1-

reactive Cys residues in Ph-B confirms these data (not shown).

This result is not surprising as the successful isolation of Ph-b

required the presence of a reductant (0.5 mM DTT), which

reduces disulfides and sulfenic acid. However, these data also

suggest that any potential oxidation of Ph-b in vivo does not

lead to a significant accumulation of Cys sulfinic and sulfonic

acid. Unfortunately, the isolation of Ph-b under non-reducing

conditions was accompanied by a significant drop in the protein

yields.

4. Discussion

This study confirms our previous observation derived from

proteomic experiments that the skeletal muscle glycogen

phosphorylase isoform PHS2 (encoded by the gene PYGM) is

a target for Tyr nitration in vivo and, furthermore, characterizes

the accumulation of 3-NT in the purified protein during

biological aging in an effort to correlate protein modification

with age-associated loss of protein function. Our earlier

proteomic study detected Ph-b nitration on Tyr113 in rat

skeletal muscle homogenate (Kanski et al., 2005b). The present

paper confirms the nitration of Tyr113 in the purified Ph-b and

presents MS/MS evidence for the in vivo nitration of Ph-b Tyr

residues at two additional locations, Tyr161 and Tyr573. Taking

into account that our MS method covered the in vitro 3-NT

formation on peptides containing 26 out of a total 35 Tyr

Fig. 5. XCorr values for 3-NT (A) and Tyr containing Ph-b peptides (B) after in vitro nitration by peroxynitrite. In (A), the asterisks at 3-NT positions 113, 161, and

573 show XCorr values for the nitropeptides detected in vivo. The XCorr values are presented mostly for the peptides with the charge state of C2 except for peptides

with (1) charge state C3 representing 3-NT at positions 113, 155, 157, 161, 511, 613, and 648 or Tyr at positions 51, 52, 113, 157, 203, 374, 553, 613, and 820, and

(2) charge state C1 representing Tyr at positions 573, 648, and 791. Peptides with the charge states C3 or C1 were normalized for the charge state C2 by

subtraction of 0.5 from or addition of 0.5, respectively, to the actual XCorr value solely to represent all data in a single plot using a single filter XCorr O2.0 (shown

by dashed lines).

V.S. Sharov et al. / Experimental Gerontology 41 (2006) 407–416414

residues (Fig. 5), this in vivo selectivity of Ph-b nitration to

three specific Tyr residues is remarkable.

Purification of Ph-b from aging rat tissue allowed not only

the localization of 3-NT to distinct sequences of the protein by

MS/MS but also quantifying the absolute amount of age-

dependent 3-NT accumulation. This quantitation is based on

both the immunoreactivity of Ph-b bands towards 3-NT-

antibodies and the HPLC–UV analysis of the purified protein.

Both methods demonstrate a statistically significant increase in

the accumulation of 3-NT in Ph-b isolated from 34 months old

rats (Figs. 2D and 3A). Moreover, quantitative HPLC

measurements provide an absolute amount of 3-NT on Ph-b

in vivo (0.05G0.03 and 0.34G0.11 mol 3-NT/mol protein for

Ph-b from 6 and 34 months old rats, respectively), which is

important for the consideration of this modification to be

responsible for the observed age-associated loss of Ph-b

function. We measured an age-dependent decrease in the

specific activity of Ph-b from 20.6G0.8 to 13.8G0.7 U mgK1

protein for 6 and 34 months old animals, respectively. Our

HPLC–UV measurements yield an age-dependent accumu-

lation of ca. 0.3 mol 3-NT per mol of Ph-b, indicating that up to

30% of the protein molecules could be modified under the

assumption that each modified molecule of Ph-b contains a

single 3-NT residue. This would lead to an excellent

correlation between modification (30%) and the observed

protein inactivation (33%). However, as the active enzyme

requires Ph-b dimer formation, a single 3-NT residue per Ph-b

dimer may be sufficient for the loss of activity. It is also known

that Tyr nitration represents only one of several potential post-

translational modifications targeting proteins under conditions

of oxidative stress, and that the functional effects of

modifications depend on their sequence location.

An important question is whether the accumulation of post-

translational modifications such as 3-NT may account for part

of the well-known loss of motor function in aging muscle

(Larsson and Ansved, 1995). First of all, proteomic studies

show that, in addition to Ph-b, several other proteins involved

in the energy metabolism of muscle are affected by age-

associated Tyr nitration (Kanski et al., 2005b) so that

synergistic effects may occur. On the other hand, even the

modification of a sequence in a single protein may cause a

serious disorder, as observed in many genetic diseases and

demonstrated by mutational analysis in animal models. In

particular, McArdle disease (myophosphorylase deficiency, or

glycogenosis type V) with a typical clinical phenotype

characterized by the progressive weakness and/or exercise

intolerance has been linked to several nonsense or missense

mutations in the PYGM gene. The most common mutation,

accounting for up to 60% alleles, is the nonsense R49X

mutation; however, 33 additional mutations have been

described (reviewed in DiMauro et al., 2002) and this number

is still growing. In contrast to the relative clinical and

biochemical homogeneity of McArdle’s disease, genetic

heterogeneity is apparent; mutations are spread throughout

V.S. Sharov et al. / Experimental Gerontology 41 (2006) 407–416 415

the gene and there is no clear genotype-phenotype correlation

(Martin et al., 2001). An important message from the genetic

studies is that, besides nonsense mutations generating

truncated protein variants (R49X, E124X, and Q754X), there

are different missense mutations of single amino acids, which

affect Ph-b function, e.g. L115P, P193W, G204S, L291P,

E348K, L396P, T487N, K542T, R601W, E654K, A659D,

Q665E, N684Y, G685R, A686P, A703V, W797R, and the

deletion of F708 (cf. SwissProt database entry P11217,

webaddress: http://ca.expasy.org/uniprot/PHS2_HUMAN).

Based on structural studies, it is well-recognized that some

Ph-b domains play a more important role in the regulation of

the protein activity, dependent on their proximity to (i) Ser14

(phosphorylation site responsible for the transformation of

homodimeric Ph-b to Ph-a, the tetrameric form of higher

specific activity); (ii) Tyr75 and, probably, Tyr155 (putative

AMP binding sites involved in the allosteric control of Ph-b

function); (iii) Cys108 and Cys142 (Involved in the association

of Ph-b homodimer subunits); and (iv) Lys680 (covalent

binding site for the cofactor pyridoxal phosphate, PLP)

(Barford et al., 1991; Titani et al., 1977). From our data, we

cannot decide, which of the three in vivo nitrated Tyr residues

may be functionally more important. Two of them may

potentially disturb local interactions within Ph-b domains

involved in the allosteric control and homodimerization due to

sequence location close to Tyr155 (for 3-NT at position 161)

and Cys108 (for 3-NT at position 113), respectively. The 3-NT

residue at position 573 is located far from functionally

important sites based on the protein sequence. However,

inspection of the 3D structure of Ph-b in the T-state PDB code:

1GPB; Barford et al., 1991) shows a close spatial proximity of

Tyr573 and Lys680-PLP (the distance between Tyr and

phosphate group amounts to only 5.7 A). Hence, the formation

of the three 3-NT residues on Ph-b may significantly affect the

enzyme activity. The vulnerability of Ph-b to Tyr nitration may

partially originate from the high abundance of Tyr residues in

the sequence (37 out of total 841 amino acids, or 4.4% for rat

muscle Ph-b). In addition, the accumulation of posttransla-

tional modifications on Ph-b may be favored by its relatively

long half-life of up to 11 days (Butler et al., 1985), and further

promoted by an even lower rate of the Ph-b turnover under

conditions of muscle dystrophy and aging (Leyland and

Beynon, 1991; Flannery et al., 1992).

Interestingly, the in vitro nitration by peroxynitrite does not

completely mimic the selectivity of nitration in vivo. In vitro,

multiple nitration sites were detected, among which was only

one out of the three nitration sites detected in vivo, Tyr113. A

similar observation was recently reported for skeletal muscle

cytosolic creatine kinase (Kanski et al., 2005b). These data

indicate the possibility of alternative nitration pathways in vivo

(Greenacre and lschiropoulos, 2001; Gow et al., 2004) and a

potentially site-specific 3-NT repair by the putative ‘denitrase’

(Irie et al., 2003). In addition, only some nitrated Ph-b isoforms

may be selected for turnover, potentially leading to the

accumulation of nitrated Ph-b isoforms, which are not subject

to protein turnover. At last, the apparent discrepancy between

ex vivo and in vitro tyrosine nitration profiles may originate

from different conformational states and/or protein interactions

that are likely to occur in vivo where Ph-b structural properties

can be affected by effector molecules and phosphorylation state

(Barford et al., 1991).

Acknowledgements

This work was supported by grants from the NIH (AG12993

and AG23551). The Q-TOF-2TM was purchased with support

from NSF EPSCoR and the University of Kansas. The Waters

CapLC and ProteinLynx Global Server were purchased for KU

by the Kansas City Area Life Sciences Institute.

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