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The human pituitary nitroproteome: detection of nitrotyrosyl-proteins

with two-dimensional Western blotting, and amino acid sequence

determination with mass spectrometryq

Xianquan Zhana, Dominic M. Desiderioa,b,c,*

a Charles B. Stout Neuroscience Mass Spectrometry Laboratory, University of Tennessee Health Science Center,

847 Monroe Avenue, Room 117, Memphis, TN 38163, USAb Department of Neurology, University of Tennessee Health Science Center, 847 Monroe Avenue, Room 117, Memphis, TN 38163, USA

c Department of Molecular Sciences, University of Tennessee Health Science Center, 847 Monroe Avenue, Room 117, Memphis, TN 38163, USA

Received 19 October 2004Available online 11 November 2004

Abstract

Nitric oxide is an important mediator that participates in reduction-oxidation (redox) mechanisms and in cellular signal transduc-tion pathways. Two types of post-translational modifications are induced by nitric oxide: S-nitrosylation of cysteine residues and nitra-tion of tyrosine residues. Two-dimensional gel electrophoresis-based Western blotting was used to detect, and liquid chromatography(LC)-tandem mass spectrometry (MS/MS) to determine the amino acid sequence of, several different nitrated proteins in the humanpituitary. Proteins from several 2D gel spots, which corresponded to the strongly positive anti-nitrotyrosine Western blot spots, weresubjected to in-gel trypsin-digestion and LC-MS/MS analysis. MS/MS, SEQUEST analysis, and de novo sequencing were used todetermine the nitration site of each nitrated peptide. A total of four different nitrated peptides were characterized and were matched

to fourdifferent proteins:synaptosomal-associated protein, actin, immunoglobulinaFc receptor,and cGMP-dependent protein kinase2. Those nitrotyrosyl-proteins participate in neurotransmission, cellular immunity, and cellular structure and mobility. 2004 Elsevier Inc. All rights reserved.

Keywords: Human pituitary; Nitration; Western blotting; Tandem mass spectrometry; De novo sequencing; Two-dimensional gel electrophoresis;Nitroproteome; Redox proteomics; Bioinformatics

Nitric oxide (NO) plays important regulatory roles inmany different biological processes such as vascularsmooth-muscle relaxation, neurotransmission, cellularimmunity, platelet aggregation, oxidative stress, cell sig-

nalling, etc.[1,2]. Nitric oxide is synthesized from LL-ar-ginine by the action of the three nitric-oxide synthase(NOS) isozymes[3]. Endothelial NOS (eNOS) and neu-ronal NOS (nNOS) isozymes have restricted tissue dis-tributions and are regulated in part by intracellularCa2+ transients. Inducible NOS (iNOS) is expressed inmany cell types in mammals after an induction by cyto-kines, lipopolysaccharide, etc., or during pathology anddiseases; once iNOS is expressed, it is active at restinglevels of intracellular Ca2+.

0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2004.10.169

q Abbreviations: ACTH, adrenocorticotropic hormone; BCIP/NBT,5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; ESI,electrospray ionization; FSH, follicle stimulating hormone; FSHRH,follicle stimulating hormone-releasing hormone; GH, growth hor-mone; IEF, isoelectic focusing; IFN, interferon; IPG, immobilized pH

gradient; LC, liquid chromatography; LH, luteinizing hormone;LHRH, luteinizing hormone-releasing hormone; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; Mr,relative molecular mass (dimensionless); MS/MS, tandem mass spec-trometry; NO, nitric oxide; NOS, nitric-oxide synthase; eNOS,endothelial NOS; nNOS, neuronal NOS; iNOS, inducible NOS;ONOO, peroxynitrite; pI, isoelectric point; PRL, prolactin; PTM,post-translational modification; Q-IT, quadrupole ion trap; SDSPAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis;2DGE, two-dimensional gel electrophoresis; TOF, time-of-flight.

* Corresponding author. Fax: +1 901 448 7842.E-mail address:[email protected](D.M. Desiderio).

www.elsevier.com/locate/ybbrc

Biochemical and Biophysical Research Communications 325 (2004) 11801186

BBRC
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Nitric oxide exerts its actions by a chemical modifica-tion of target molecules by preferentially interactingwith thiol groups (such as in cysteine), transition metals(Men+; Me = metal), free radicals (such as the superox-ide anion), key redox regulators (such as glutathione),etc. The NO-related chemical relationships with other

molecules have been reviewed [4]. The S-nitrosylationof cysteine residues and the nitration of tyrosine residuesin target proteins have been studied in redox-based post-translational modifications (PTMs). S-nitrosylation isthe addition of NO to a sulfur atom by forming an SNO bond. S-nitrosylation is emerging as a ubiquitous,specific, and reversible regulatory mechanism, similarto phosphorylaton, in cellular signal transduction path-ways[4,5].S-nitrosylated proteins often serve as the ma-jor effectors of NO-related bioactivity [3]. Nitration is,generally, the addition of a nitro (NO2) group to posi-tion 3 of the phenolic ring of a tyrosine residue, andclearly is a consequence of the formation of peroxyni-

trite (ONOO) in a cell. Tyrosine nitration is one of sev-eral different protein modifications that result fromoxidative stress[6], and results in the alteration of pro-tein function. NOS isoenzymes provide the biologicalprecursor for the in vivo nitrating agents that performthat modification. Many neurodegenerative and inflam-matory diseases have been demonstrated to be associ-ated with the nitration of tyrosine [7,8]; for example,cancer, Parkinsons disease, Alzheimers disease, Hun-tingtons disease, lung infection, retinal ischemia, etc.

The human pituitary is the master regulatory gland inthe multiple hypothalamicpituitarytarget organ axes

systems, and plays important roles in many differentphysiology and pathology processes. Many studies haveindicated the presence of NOS in the human and ratpituitary[913], and gonadotrophs and folliculostellatecells have been reported to contain NOS. The eNOS,nNOS, and iNOS isoenzymes are expressed in the pitu-itary gland and in pituitary adenomas, and an increasedactivity of eNOS has been found in the endothelial cellsof pituitary adenomas. The nNOS isoenzyme and itsmRNA were also found to be increased in human pitu-itary adenomas, and were located to the secretory andfolliculostellate cells. The iNOS was also present in ratanterior pituitary cells that had been induced byinterferon (IFN)-c, which increased NO production[14]. NO plays an important role in activating the releaseof luteinizing hormone-releasing hormone (LHRH)and follicle-stimulating hormone-releasing hormone(FSHRH) from the hypothalamus, and of LH andFSH from the pituitary[1518]. Moreover, the NO in-volved in LH secretion either originates in gonadotrophsor it requires the participation of gonadotrophs[9]. NOmay either stimulate or inhibit the secretion of PRL[1921]. The circulating levels of NO change in dopamine-treated hyperprolactinoma patients [22]. NO regulatesthe secretion of growth hormone (GH) in the normal

human pituitary and in acromegaly[11,23,24], and mod-ulates GH secretion in a dose-dependent manner in GHadenomatous cells from human pituitary adenomas[25].NO also plays an important role in the control of thehypothalamicpituitaryadrenocortical axis [26]. NOinhibits the release of ACTH; the adipocyte hormone

leptin, a member of the cytokine family, has largelyopposite actions to those of the proinflammatory cyto-kines [18]. Although much progress has been made inclarifying the role of NO in the human pituitary andpituitary adenomas, little is known about the proteintargets of NO-mediated PTMs in the human pituitaryand in pituitary tumors.

In this study, we investigated the presence of, and thepotential roles of, the nitration of tyrosine-containingproteins in the normal (control) human pituitary.Anti-nitrotyrosine antibodies were used to detect nitro-tyrosyl-proteins on a PVDF membrane after the pro-teins were transferred from the two-dimensional gel,

and liquid chromatography-tandem mass spectrometrywas used to determine the amino acid sequence of thosenitrotyrosyl-proteins. Each amino acid sequence andnitration site was determined with SEQUEST analysisand de novo sequencing. A total of four nitrotyrosyl-proteins were characterized in five immunoreactive-posi-tive 2D gel spots. These results provide a platform toinvestigate the nitroproteome in the human pituitary,and to explore the potential physiological roles of pro-tein nitration in the normal human pituitary.

Materials and methods

Pituitary tissue and extraction of proteins. A normal (control)human pituitary tissue post-mortem sample (male, 45 years-old,drowning) was obtained from the Memphis Regional Medical Center.The processing of tissue and the extraction of proteins were carried outaccording to our previous procedure[27].

Two-dimensional gel electrophoresis and protein staining. Firstdimensionisoelectric focusing (IEF) was performed with anAmersham Multiphor II instrument with protein (70 lg; 360 ll of theextracted protein sample solution) loaded onto an 18-cm IPGstrip(pH 3-10 NL, AmershamPharmacia Biotech, Piscataway, NJ, USA).After equilibration with the solution that contained iodoacetamide,the IEF-separated proteins, second-dimensionsodium dodecyl sul-

fatepolyacrylamide gel electrophoresis (SDSPAGE) was performedwith a home-made 12% PAGE resolving gel (190 205 1 mm) on avertical PROTEAN-plus Dodeca Cell (Bio-Rad, Hercules, CA,USA). The two-dimensional gel electrophoresis (2DGE)-separatedprotein spots were visualized with a modified silver-staining methodthat is compatible with MALDI-TOF-MS. Those methods were de-scribed elsewhere in greater detail [27].

Western blotting. For immunoblotting analysis, the 2DGE wasperformed in the same way as described above. The proteins in the 2Dgel were transferred to a PVDF membrane (0.8 mA/cm2; 1 h, 40 min)with a Pharmacia Biotech Nova Blot semi-dry transfer instrument. ThePVDF membrane that now contained the proteins was blocked(30 min) with a solution (100 ml) of 0.3% bovine serum albumin/phosphate-buffered saline (BSA/PBS) with 0.1% sodium azide and0.2% Tween 20 (PBST). The BSA-blocked PVDF membrane was

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incubated (1 h) with a rabbit anti-human nitrotyrosine antibody(Sigma, N0409, St. Louis, MO, USA) that was diluted (1:1000 = v:v)in a 0.3% BSA/PBST solution. After completion of the incubation withthe primary antibody, the membrane was washed with the PBSTsolution (100 ml; 15 min 3). The secondary antibody [goat anti-rab-bit alkaline phosphase-conjugated IgG (Pierce, Rockford, IL, USA),diluted (1:5000 = v:v)] in a 0.3% BSA/PBST solution, was added to theblots (1 h, room temperature). The membrane was washed with PBST(100 ml; 15 min 3), and nitroproteins were visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT)(Pierce, Rockford, IL, USA). A parallel negative-control experimentwas carried out in order to observe any cross-reactivity of the sec-ondary antibody. For the negative-control experiment (the primaryantibody was not added), the entire procedure was the same as theWestern blotting. The 2DGE gel, after transferring proteins to PVDFmembrane, was silver-stained in the same way as described above inorder to detect any proteins that might have remained on the gel and todetermine the efficiency of the protein transfer.

Image analyses of a 2DGE gel and of a Western blot membrane. Thescanned images of the silver-stained 2D gels and of the visualizedWestern blot membranes were input to a PDQuest system (Bio-Rad,

version 7.1, Hercules, CA) to generate the synthetic image that con-tained the Gaussian spots (Gaussian image) with a defined volume[volume = optical density (OD) width (mm) length (mm)] andquality [28]. All subsequent spot-matching and analysis steps wereperformed on the Gaussian spots. In order to minimize the effect ofany experimental factor on a spot volume, each spot volume wasnormalized to the total optical density in each gel image[28].

Mass spectrometry characterization of nitrated proteins. The silver-stained 2D gel-spots (labeled inFig. 1A; spots 14, 8, 9, 14, and 15)that corresponded to the positive Western blot spots were excised, andthe proteins were subjected to in-gel trypsin digestion[27]. The tryptic-peptide mixture was purified with a ZipTipC18 micro-columnaccording to the manufacturers instructions. The purified trypticpeptide mixture was analyzed with a capillary liquid chromatography-LCQDeca mass spectrometer (LC-ESI-Q-IT) equipped with a standardelectrospray ionization (ESI) source (ThermoFinnigan, San Jose, CA,USA) to obtain the amino acid sequence of each peptide. Those de-tailed experimental methods have been described elsewhere [27].

Determination of nitration sites. De novo sequencing was used toindependently and accurately determine each amino acid sequence.The amino acid sequence from de novo sequencing was used to search

Fig. 1. Two-dimensional Western blot analysis of anti-3-nitrotyrosine proteins in a human pituitary (70 lg protein per 2D gel). (A) Silver-stainedimage on a 2D gel before transfer of proteins to a PVDF membrane. (B) Silver-stained image on a 2D gel after transfer of proteins to a PVDFmembrane. (C) Western blot image of anti-3-nitrotyrosine proteins (anti-3-nitrotyrosine antibodies + secondary antibody). (D) Negative control ofWestern blot to show the cross-reaction of the secondary antibody (only the secondary antibody; no anti-3-nitrotyrosine antibody).

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human SWISS-PROT protein database with the SIB BLAST searchengine (http://us.expasy.org/tools/blast/). The LC-ESI-Q-IT MS/MSdata were used to identify the protein by searching the SWISS-PROTand NCBInr databases with the SEQUEST software that is a part ofthe LCQDeca software package. Mass modifications of +45 kDa (+NO2H) at Tyr and of +57 kDa (+NH2COCH2H) at Cys wereconsidered in the search. Each positive search result nitration of aTyr residue- was confirmed with a manual check of the original LC,MS, and MS/MS data to determine each nitration site. During theanalysis of those nitrated proteins, the following experimental criteriawere applied: K or R at the C-terminus; K, R, or D [29]preceding theN-terminus; 0 or 1 missed trypsin cleavage sites; singly chargedproduct b- and y-ions, and Homo sapiens.

Results

Two-dimensional gel electrophoresis-based Western blot

detection of nitrotyrosyl-proteins

Ca. 1000 protein spots were detected in each silver-

stained 2D gel[27,30]. Those positive-stained nitrotyro-sine-containing proteins that were transferred onto aPVDF membrane were detected with an anti-3-nitroty-rosine antibody (Fig. 1). Each 2D gel in Fig. 1 encom-passes the region pI with 4.36.0 and Mr 1860 kDa.In order to determine any cross-reactivity of the second-ary antibody with a protein, parallel negative-controlexperiments were performed. Fig. 1A shows the silver-stained 2D gel spots before the proteins were transferredonto a PVDF membrane. Fig. 1B is the correspondingsilver-stained 2D gel image after the proteins were trans-ferred onto the PVDF membrane, and demonstrates

that proteins were completely transferred onto thePVDF membrane. Fig. 1C is the Western blot imagewith positive nitrotyrosine-immunoreactivity; 16 Wes-tern blot spots were detected.

Four spots (spots 14) demonstrated a high level ofnitrotyrosine-immunoreactivity. Non-nitro-Tyr-proteinsalso existed in those four spots (spots 14) (but withmuch lower intensity) in the negative control (Fig.1D). The intensity of the spots of the Western blot(W) spots (Fig. 1C) and the corresponding negative con-trol (N) (Fig. 1D) were semi-quantified, and those W/Nratios are given inTable 1.Those results show that theintensity of each one of those four Western blot spots

was much higher than that of the corresponding nega-tive-control spot. Those data suggested that nitrotyro-syl-proteins were contained in those four spots, and byextension also in the other spots in Figs. 1A and C.

LC-MS/MS determination of nitrotyrosyl-proteins

MS/MS data, SEQUEST software, and de novosequencing were used to obtain the amino acid sequenceof each nitrated peptide, and to locate the nitration sitein each nitrotyrosyl-protein. Eight strongly stained (sil-ver) spots inFig. 1A (spots 14, 8, 9, 14, and 15) thatcorresponded to the strongly positive Western blot spotsin Fig. 1C were excised, and were subjected to in-geltrypsin digestion and LC-ESI-MS/MS analysis. A totalof four different nitrotyrosyl-peptides were sequencedin five spots (1, 2, 4, 14, and 15), and those sequenceswere matched to four different proteins. Those fournitrotyrosyl-peptides and the corresponding four pro-

teins are listed inTable 2, which contains the spot num-ber, the name and SWISS-PROT number of eachprotein, the amino acid sequence of each nitrotyrosyl-peptide, the location of each nitrotyrosine residue, theSEQUEST Xcorr scores, and the sequencing method(SEQUEST; de novo).

A representative LC-MS/MS analysis of a nitrotyro-syl-peptide that contained 11 amino acids is shownin Fig. 2. A doubly charged ion ([M + 2H]2+,m/z= 686.12) in the MS spectrum (scan num-ber = 2177) of the peptide that eluted at retention time(RT) = 52.22 min was selected after a SEQUEST analy-

sis. Fig. 2 contains the MS/MS spectra of that[M + 2H]2+ precursor ion. The product ions labeled inFig. 2(top right) derived from the SEQUEST analysisand include the singly charged b- and y-ions. The corre-sponding amino acid sequence 228GQC#KDALEI*YK238 (*Y, nitrated Tyr, C#, Cys_CAM) is alsoshown. That nitrotyrosyl-peptide matched sequence228238 of the synaptosomal-associated protein(SWISS-PTOT number = O60641) (Table 2). A nitroty-rosine was assigned to Tyr-237. Moreover, the loss ofH2O from the singly charged b4 ion, and the loss ofNH3 from the singly charged b8 and b10 ions were de-tected. The MS/MS spectrum was also analyzed de novo(bottom) (Fig. 2). The de novo sequence (KDALEIYK)was matched to the sequence 231238 of the same pro-tein (Swiss-Prot number = O60641) with BLAST analy-sis; and a mass difference +45 (+NO2H) was assignedto the residue Tyr-237. The SEQUEST sequence wasvalidated by the more accurate de novo analysis.

Functional characteristics of the characterized

nitrotyrosyl-proteins

PTM analysis is an important factor in proteomicsbecause a given PTM analysis of the specific functional

Table 1Semi-quantitative analysis of the Western blot spot volume(ratio = W/N)

Spot number Test 1 Test 2 xn 2

1 4.9 55.9 30.42 2.7 3.4 3.13 5.3 14.2 9.84 3.9 4.5 4.2

W, western blotting; N, negative control. Tests 1 and 2 refer to sepa-rate experiments. The transfer time was 1 h, 45 min for tests 1 and 2.The ratio of the OD of W to N is given.

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proteome could reveal the roles and potential functionsof each PTM in human physiology and disease states.NO is an important mediator in the redox processesand cell-signal transduction. An interesting phenome-

non is that, among those four characterized nitrotyro-syl-proteins in the human pituitary, synaptosomal-associated protein, and cGMP-dependent protein kinase2 significantly correlated to the cellular signal pathways;

Table 2Human pituitary nitrated proteins determined by amino acid sequencing (SEQUEST; de novo) from liquid chromatography-tandem massspectrometry

Spotnumber

Protein name Swiss-Prot number Nitrotyrosyl-peptide Tyrnitrationsite

SequestXcorr

Interpretation

1 Synaptosomal-associated

protein

O60641 228(K)GQC#KDALEI*YK238 237 3.44 SEQUEST

231KDALEI*YK238 237 de novo2 Immunoglobulin a Fc

receptorP24071 223(D)*YTTQNLIR230 223 1.87 SEQUEST

223*YTTQNLIR230 223 de novo4 Immunoglobulin a Fc

receptorP24071 223(D)*YTTQNLIR230 223 1.95 SEQUEST

223*TTQNLIR230 223 de novo14 Actin P03996 or 294(K)DL*YANNVLSGGTTMYPGIADR314 296 3.25 SEQUEST

P12718 or 293(K)DL*YANNVLSGGTTMYPGIADR313 295P04270 294(K)DL*YANNVLSGGTTMYPGIADR314 296

15 cGMP-dependent proteinkinase 2

Q13237 352(K)GE*YFGEKALI361 354 1.96 SEQUEST

352GE*YFGEK358 354 de novo

*Y, nitrotyrosine; C#

, Cys-CAM. The bracket refers to the amino acid residue that preceded the N-terminus of the nitrated peptide.

Fig. 2. SEQUEST (top-right) and de novo (bottom) analysis of an MS2 spectrum of the precursor ion ([M + 2H]2+, at m/z= 686.12,RT = 52.30 min, and scan number 2180) for a nitrotyrosyl peptide (Tyr-237) 228GQC#KDALEI*YK238 that contained 11 amino acids and thatderived from synaptosomal-associated protein (spot 1).



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immunoglobulin a Fc receptor participates in the cellu-lar immunity; and actin is an important member in cel-lular structure and mobility. With this describedmethodology to detect and to MS-characterize proteins,more nitrotyrosyl-proteins, especially low-abundancenitrotyrosyl-proteins, will be identified to comprehen-

sively reveal the presence of, and the roles of, nitricoxide-induced nitrotrysosyl modification in the humanpituitary.

Discussion

In this human pituitary study, the proteins in eight(Fig. 1A; spots 14, 8, 9, 14, and 15) strongly positiveanti-3-nitrotyrosine Western blot 2D gel spots were ana-lyzed with LC-MS/MS. The amino acid sequences offour different nitrotyrosyl-peptides in five 2D gel spots(spots 1, 2, 4, 14, and 15) were determined with MS/

MS sequencing (de novo; SEQUEST) and were matchedto four different proteins. Those four nitrated proteinsparticipate in important physiology process, includingneurotransmission, cellular immunity, and cellularstructure and mobility; and might provide potentialmolecular clues to further study the role of NO-inducedPTMs in the human pituitary.

The human genome contains ca. 30,000 genes thatcode ca. 100,000 proteins. Most of the proteins distrib-ute on a 2D gel within the area of pH 310. Here,2DGE was performed with 18-cm IPG strip pH 310NL. For the pituitary proteome, a 2DGE gel can detect

ca. 1000 spots[27,30]. We used LC-MS/MS to analyzethe proteins contained within those spots; each spot con-tained several proteins. In addition to nitrotyrosyl-pro-teins, high-abundance human growth hormones werealso contained in each spot (spots 14, 8, and 9), othertypes of actin cytoplasmic (SWISS-PROT numberP02570 and P02571) in spot 14, and tubulin beta in spot16, which is consistent with our previous results [30,31].Other studies also found that one 2D gel spot could in-clude several proteins (six proteins) [32].

The accurate de novo sequencing method, and theless-accurate SEQUEST spectra-comparison method,are two different methods to obtain the amino acid se-quence of a peptide that had been analyzed in the MS/MS mode. The basic procedure of SEQUEST analysisis that, for each peptide from a protein in the database,the list of all singly charged ions m/z values and alldoubly charged ions m/z values is calculated. The ob-served m/z values in the product-ion spectrum of a pre-cursor ion are matched and correlated, according to aspecial statistical model, to those theoretical m/z values.Some indices (e.g., Xcorr, dCn, etc.) are calculated toprovide a parameter that represents a goodness-of-fitbetween the theoretical and the measured spectra.The best-fit data represent the peptide sequence. For

de novo sequencing, the observed m/z values in theproduct-ion spectrum of a precursor ion are used tomanually deduce the amino acid sequence from the dif-ference between the m/z values of abundant ions. Eachamino acid sequence from each method was used tomatch the protein sequence in a database to determine

the protein. For an MS/MS spectrum from a precursorion, the peptide sequence length derived from SE-QUEST analysis might be longer than that sequencefrom de novo sequencing; for example, for spots 1and 15 (Table 2). De novo sequencing more accuratelydetermines an amino sequence and the assignment of aTyr nitration site; especially for a peptide with a non-significant SEQUEST Xcorr score, and/or no K/R atthe C-terminus, and no K/R preceding the N-terminus.For example, the amino acid residue that precedes theN-terminus of *YTTQNLIR is the acid-labile D resi-due [29] (Table 2); the purified tryptic peptides werekept in an acidic solution. The amino acid residue at

the C-terminus of GE*YFGEKALI is the residue I(Table 2); that peptide might have been produced be-cause no protease inhibitors were used to extract pro-teins; for example, some endogenous proteinases maycleave residue I at its C-terminus (http://us.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html).De novo analysis can almost always provide moreaccurate amino acid sequence than SEQUESTanalysis.

The integration of Western blotting and LC-MS/MSis an effective approach to detect and characterize nitro-tyrosyl-protein in the human pituitary proteome. Two-

dimensional gel electrophoresis-based Western blottingcan pre-separate and enrich proteins with a similar pIand Mr. LC can real-time pre-separate and enrich thosetryptic peptides before mass spectrometry analysis. MS/MS can accurately locate each nitration site. This meth-odology provides a basis to comprehensively investigatethe nitroproteome in the human pituitary, especially toachieve our goal to detect and characterize pituitaryadenoma-related nitrotyrosyl-proteins in a program toclarify the basic molecular mechanisms of pituitary ade-noma formation.

Acknowledgments

The authors gratefully acknowledge financial assis-tance (to D.M.D.) from the National Institutes ofHealth (NS 42843). The MALDI-TOF mass spec-trometer was purchased with grants (D.M.D.) fromNIH (RR-10522) and NSF (DBI 9604633), and theLCQ with a grant (D.M.D.) from NIH (RR-14593).We acknowledge the helpful discussions with CliveSlaughter. The pituitary control tissues were providedby the Memphis Regional Medical Center (Memphis,TN, USA).

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