BBRC

Biochemical and Biophysical Research Communications 325 (2004) 11801186 www.elsevier.com/locate/ybbrc

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,*
Charles B. Stout Neuroscience Mass Spectrometry Laboratory, University of Tennessee Health Science Center, 847 Monroe Avenue, Room 117, Memphis, TN 38163, USA b Department of Neurology, University of Tennessee Health Science Center, 847 Monroe Avenue, Room 117, Memphis, TN 38163, USA Department of Molecular Sciences, University of Tennessee Health Science Center, 847 Monroe Avenue, Room 117, Memphis, TN 38163, USA Received 19 October 2004 Available online 11 November 2004
a

Abstract Nitric oxide is an important mediator that participates in reduction-oxidation (redox) mechanisms and in cellular signal transduction pathways. Two types of post-translational modications are induced by nitric oxide: S-nitrosylation of cysteine residues and nitration 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 dierent nitrated proteins in the human pituitary. Proteins from several 2D gel spots, which corresponded to the strongly positive anti-nitrotyrosine Western blot spots, were subjected to in-gel trypsin-digestion and LC-MS/MS analysis. MS/MS, SEQUEST analysis, and de novo sequencing were used to determine the nitration site of each nitrated peptide. A total of four dierent nitrated peptides were characterized and were matched to four dierent proteins: synaptosomal-associated protein, actin, immunoglobulin a Fc receptor, and cGMP-dependent protein kinase 2. 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

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 hormone; IEF, isoelectic focusing; IFN, interferon; IPG, immobilized pH gradient; LC, liquid chromatography; LH, luteinizing hormone; LHRH, luteinizing hormone-releasing hormone; MALDI, matrixassisted laser desorption/ionization; MS, mass spectrometry; Mr, relative molecular mass (dimensionless); MS/MS, tandem mass spectrometry; 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 modication; Q-IT, quadrupole ion trap; SDS PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; 2DGE, two-dimensional gel electrophoresis; TOF, time-of-ight. * Corresponding author. Fax: +1 901 448 7842. E-mail address: ddesiderio@utmem.edu (D.M. Desiderio).

Nitric oxide (NO) plays important regulatory roles in many dierent biological processes such as vascular smooth-muscle relaxation, neurotransmission, cellular immunity, platelet aggregation, oxidative stress, cell signalling, etc. [1,2]. Nitric oxide is synthesized from L -arginine by the action of the three nitric-oxide synthase (NOS) isozymes [3]. Endothelial NOS (eNOS) and neuronal NOS (nNOS) isozymes have restricted tissue distributions and are regulated in part by intracellular Ca2+ transients. Inducible NOS (iNOS) is expressed in many cell types in mammals after an induction by cytokines, lipopolysaccharide, etc., or during pathology and diseases; once iNOS is expressed, it is active at resting levels of intracellular Ca2+.

0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.10.169

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Nitric oxide exerts its actions by a chemical modication of target molecules by preferentially interacting with thiol groups (such as in cysteine), transition metals (Men+; Me = metal), free radicals (such as the superoxide anion), key redox regulators (such as glutathione), etc. The NO-related chemical relationships with other molecules have been reviewed [4]. The S-nitrosylation of cysteine residues and the nitration of tyrosine residues in target proteins have been studied in redox-based posttranslational modications (PTMs). S-nitrosylation is the addition of NO to a sulfur atom by forming an S NO bond. S-nitrosylation is emerging as a ubiquitous, specic, and reversible regulatory mechanism, similar to phosphorylaton, in cellular signal transduction pathways [4,5]. S-nitrosylated proteins often serve as the major eectors of NO-related bioactivity [3]. Nitration is, generally, the addition of a nitro (NO2) group to position 3 of the phenolic ring of a tyrosine residue, and clearly is a consequence of the formation of peroxynitrite (ONOO) in a cell. Tyrosine nitration is one of several dierent protein modications that result from oxidative stress [6], and results in the alteration of protein function. NOS isoenzymes provide the biological precursor for the in vivo nitrating agents that perform that modication. Many neurodegenerative and inammatory diseases have been demonstrated to be associated with the nitration of tyrosine [7,8]; for example, cancer, Parkinsons disease, Alzheimers disease, Huntingtons disease, lung infection, retinal ischemia, etc. The human pituitary is the master regulatory gland in the multiple hypothalamicpituitarytarget organ axes systems, and plays important roles in many dierent physiology and pathology processes. Many studies have indicated the presence of NOS in the human and rat pituitary [913], and gonadotrophs and folliculostellate cells have been reported to contain NOS. The eNOS, nNOS, and iNOS isoenzymes are expressed in the pituitary gland and in pituitary adenomas, and an increased activity of eNOS has been found in the endothelial cells of pituitary adenomas. The nNOS isoenzyme and its mRNA were also found to be increased in human pituitary adenomas, and were located to the secretory and folliculostellate cells. The iNOS was also present in rat anterior pituitary cells that had been induced by interferon (IFN)-c, which increased NO production [14]. NO plays an important role in activating the release of luteinizing hormone-releasing hormone (LHRH) and follicle-stimulating hormone-releasing hormone (FSHRH) from the hypothalamus, and of LH and FSH from the pituitary [1518]. Moreover, the NO involved in LH secretion either originates in gonadotrophs or it requires the participation of gonadotrophs [9]. NO may either stimulate or inhibit the secretion of PRL [19 21]. The circulating levels of NO change in dopaminetreated hyperprolactinoma patients [22]. NO regulates the secretion of growth hormone (GH) in the normal

human pituitary and in acromegaly [11,23,24], and modulates GH secretion in a dose-dependent manner in GH adenomatous cells from human pituitary adenomas [25]. NO also plays an important role in the control of the hypothalamicpituitaryadrenocortical axis [26]. NO inhibits the release of ACTH; the adipocyte hormone leptin, a member of the cytokine family, has largely opposite actions to those of the proinammatory cytokines [18]. Although much progress has been made in clarifying the role of NO in the human pituitary and pituitary adenomas, little is known about the protein targets of NO-mediated PTMs in the human pituitary and in pituitary tumors. In this study, we investigated the presence of, and the potential roles of, the nitration of tyrosine-containing proteins in the normal (control) human pituitary. Anti-nitrotyrosine antibodies were used to detect nitrotyrosyl-proteins on a PVDF membrane after the proteins were transferred from the two-dimensional gel, and liquid chromatography-tandem mass spectrometry was used to determine the amino acid sequence of those nitrotyrosyl-proteins. Each amino acid sequence and nitration site was determined with SEQUEST analysis and de novo sequencing. A total of four nitrotyrosylproteins were characterized in ve immunoreactive-positive 2D gel spots. These results provide a platform to investigate the nitroproteome in the human pituitary, and to explore the potential physiological roles of protein 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 out according to our previous procedure [27]. Two-dimensional gel electrophoresis and protein staining. First dimensionisoelectric focusing (IEF) was performed with an Amersham Multiphor II instrument with protein (70 lg; 360 ll of the extracted 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 sulfatepolyacrylamide gel electrophoresis (SDSPAGE) was performed with a home-made 12% PAGE resolving gel (190 205 1 mm) on a vertical PROTEAN-plus Dodeca Cell (Bio-Rad, Hercules, CA, USA). The two-dimensional gel electrophoresis (2DGE)-separated protein spots were visualized with a modied silver-staining method that is compatible with MALDI-TOF-MS. Those methods were described elsewhere in greater detail [27]. Western blotting. For immunoblotting analysis, the 2DGE was performed in the same way as described above. The proteins in the 2D gel were transferred to a PVDF membrane (0.8 mA/cm2; 1 h, 40 min) with a Pharmacia Biotech Nova Blot semi-dry transfer instrument. The PVDF membrane that now contained the proteins was blocked (30 min) with a solution (100 ml) of 0.3% bovine serum albumin/ phosphate-buered saline (BSA/PBS) with 0.1% sodium azide and 0.2% Tween 20 (PBST). The BSA-blocked PVDF membrane was

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X. Zhan, D.M. Desiderio / Biochemical and Biophysical Research Communications 325 (2004) 11801186 version 7.1, Hercules, CA) to generate the synthetic image that contained the Gaussian spots (Gaussian image) with a dened volume [volume = optical density (OD) width (mm) length (mm)] and quality [28]. All subsequent spot-matching and analysis steps were performed on the Gaussian spots. In order to minimize the eect of any experimental factor on a spot volume, each spot volume was normalized to the total optical density in each gel image [28]. Mass spectrometry characterization of nitrated proteins. The silverstained 2D gel-spots (labeled in Fig. 1A; spots 14, 8, 9, 14, and 15) that corresponded to the positive Western blot spots were excised, and the proteins were subjected to in-gel trypsin digestion [27]. The trypticpeptide mixture was puried with a ZipTipC18 micro-column according to the manufacturers instructions. The puried tryptic peptide mixture was analyzed with a capillary liquid chromatographyLCQDeca mass spectrometer (LC-ESI-Q-IT) equipped with a standard electrospray ionization (ESI) source (ThermoFinnigan, San Jose, CA, USA) to obtain the amino acid sequence of each peptide. Those detailed experimental methods have been described elsewhere [27]. Determination of nitration sites. De novo sequencing was used to independently and accurately determine each amino acid sequence. The amino acid sequence from de novo sequencing was used to search

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 with the primary antibody, the membrane was washed with the PBST solution (100 ml; 15 min 3). The secondary antibody [goat anti-rabbit alkaline phosphase-conjugated IgG (Pierce, Rockford, IL, USA), diluted (1:5000 = v:v)] in a 0.3% BSA/PBST solution, was added to the blots (1 h, room temperature). The membrane was washed with PBST (100 ml; 15 min 3), and nitroproteins were visualized with 5-bromo4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) (Pierce, Rockford, IL, USA). A parallel negative-control experiment was carried out in order to observe any cross-reactivity of the secondary antibody. For the negative-control experiment (the primary antibody was not added), the entire procedure was the same as the Western blotting. The 2DGE gel, after transferring proteins to PVDF membrane, was silver-stained in the same way as described above in order to detect any proteins that might have remained on the gel and to determine the eciency of the protein transfer. Image analyses of a 2DGE gel and of a Western blot membrane. The scanned images of the silver-stained 2D gels and of the visualized Western blot membranes were input to a PDQuest system (Bio-Rad,

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

X. Zhan, D.M. Desiderio / Biochemical and Biophysical Research Communications 325 (2004) 11801186 human SWISS-PROT protein database with the SIB BLAST search engine (http://us.expasy.org/tools/blast/). The LC-ESI-Q-IT MS/MS data were used to identify the protein by searching the SWISS-PROT and NCBInr databases with the SEQUEST software that is a part of the LCQDeca software package. Mass modications of +45 kDa (+ NO2H) at Tyr and of +57 kDa (+NH2COCH2H) at Cys were considered in the search. Each positive search result nitration of a Tyr residue- was conrmed with a manual check of the original LC, MS, and MS/MS data to determine each nitration site. During the analysis of those nitrated proteins, the following experimental criteria were applied: K or R at the C-terminus; K, R, or D [29] preceding the N-terminus; 0 or 1 missed trypsin cleavage sites; singly charged product b- and y-ions, and Homo sapiens.

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was much higher than that of the corresponding negative-control spot. Those data suggested that nitrotyrosyl-proteins were contained in those four spots, and by extension also in the other spots in Figs. 1A and C. LC-MS/MS determination of nitrotyrosyl-proteins MS/MS data, SEQUEST software, and de novo sequencing were used to obtain the amino acid sequence of each nitrated peptide, and to locate the nitration site in each nitrotyrosyl-protein. Eight strongly stained (silver) spots in Fig. 1A (spots 14, 8, 9, 14, and 15) that corresponded to the strongly positive Western blot spots in Fig. 1C were excised, and were subjected to in-gel trypsin digestion and LC-ESI-MS/MS analysis. A total of four dierent nitrotyrosyl-peptides were sequenced in ve spots (1, 2, 4, 14, and 15), and those sequences were matched to four dierent proteins. Those four nitrotyrosyl-peptides and the corresponding four proteins are listed in Table 2, which contains the spot number, the name and SWISS-PROT number of each protein, the amino acid sequence of each nitrotyrosylpeptide, the location of each nitrotyrosine residue, the SEQUEST Xcorr scores, and the sequencing method (SEQUEST; de novo). A representative LC-MS/MS analysis of a nitrotyrosyl-peptide that contained 11 amino acids is shown in Fig. 2. A doubly charged ion ([M + 2H]2+, m/z = 686.12) in the MS spectrum (scan number = 2177) of the peptide that eluted at retention time (RT) = 52.22 min was selected after a SEQUEST analysis. Fig. 2 contains the MS/MS spectra of that [M + 2H]2+ precursor ion. The product ions labeled in Fig. 2 (top right) derived from the SEQUEST analysis and include the singly charged b- and y-ions. The corresponding amino acid sequence 228GQC#KDA LEI*YK238 (*Y, nitrated Tyr, C#, Cys_CAM) is also shown. That nitrotyrosyl-peptide matched sequence 228238 of the synaptosomal-associated protein (SWISS-PTOT number = O60641) (Table 2). A nitrotyrosine was assigned to Tyr-237. Moreover, the loss of H2O from the singly charged b4 ion, and the loss of NH3 from the singly charged b8 and b10 ions were detected. 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 protein (Swiss-Prot number = O60641) with BLAST analysis; and a mass dierence +45 (+NO2H) was assigned to the residue Tyr-237. The SEQUEST sequence was validated by the more accurate de novo analysis. Functional characteristics of the characterized nitrotyrosyl-proteins PTM analysis is an important factor in proteomics because a given PTM analysis of the specic functional

Results Two-dimensional gel electrophoresis-based Western blot detection of nitrotyrosyl-proteins Ca. 1000 protein spots were detected in each silverstained 2D gel [27,30]. Those positive-stained nitrotyrosine-containing proteins that were transferred onto a PVDF membrane were detected with an anti-3-nitrotyrosine antibody (Fig. 1). Each 2D gel in Fig. 1 encompasses the region pI with 4.36.0 and Mr 1860 kDa. In order to determine any cross-reactivity of the secondary antibody with a protein, parallel negative-control experiments were performed. Fig. 1A shows the silverstained 2D gel spots before the proteins were transferred onto a PVDF membrane. Fig. 1B is the corresponding silver-stained 2D gel image after the proteins were transferred onto the PVDF membrane, and demonstrates that proteins were completely transferred onto the PVDF membrane. Fig. 1C is the Western blot image with positive nitrotyrosine-immunoreactivity; 16 Western blot spots were detected. Four spots (spots 14) demonstrated a high level of nitrotyrosine-immunoreactivity. Non-nitro-Tyr-proteins also existed in those four spots (spots 14) (but with much 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 control (N) (Fig. 1D) were semi-quantied, and those W/N ratios are given in Table 1. Those results show that the intensity of each one of those four Western blot spots

Table 1 Semi-quantitative analysis of the Western blot spot volume (ratio = W/N) Spot number 1 2 3 4 Test 1 4.9 2.7 5.3 3.9 Test 2 55.9 3.4 14.2 4.5  x n 2 30.4 3.1 9.8 4.2

W, western blotting; N, negative control. Tests 1 and 2 refer to separate 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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Table 2 Human pituitary nitrated proteins determined by amino acid sequencing (SEQUEST; de novo) from liquid chromatography-tandem mass spectrometry Spot number 1 Protein name Swiss-Prot number Nitrotyrosyl-peptide Tyr nitration site 237 237 223 223 223 223 296 295 296 354 354 Sequest Xcorr 3.44 Interpretation

Synaptosomal-associated protein Immunoglobulin a Fc receptor Immunoglobulin a Fc receptor Actin

O60641

228

(K)GQC#KDALEI*YK238 KDALEI*YK238 (D)*YTTQNLIR230 *YTTQNLIR230 (D)*YTTQNLIR230

SEQUEST de novo SEQUEST de novo SEQUEST de novo SEQUEST

231

P24071

223

1.87

223

P24071

223

1.95

223

14

15

cGMP-dependent protein kinase 2

#

P03996 or P12718 or P04270 Q13237

*TTQNLIR230 (K)DL*YANNVLSGGTTMYPGIADR314 293 (K)DL*YANNVLSGGTTMYPGIADR313 294 (K)DL*YANNVLSGGTTMYPGIADR314 352 (K)GE*YFGEKALI361

294 352

3.25

1.96

SEQUEST de novo

GE*YFGEK358

*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 that derived from synaptosomal-associated protein (spot 1).

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

non is that, among those four characterized nitrotyrosyl-proteins in the human pituitary, synaptosomalassociated protein, and cGMP-dependent protein kinase 2 signicantly correlated to the cellular signal pathways;

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immunoglobulin a Fc receptor participates in the cellular immunity; and actin is an important member in cellular structure and mobility. With this described methodology to detect and to MS-characterize proteins, more nitrotyrosyl-proteins, especially low-abundance nitrotyrosyl-proteins, will be identied to comprehensively reveal the presence of, and the roles of, nitric oxide-induced nitrotrysosyl modication in the human pituitary.

Discussion In this human pituitary study, the proteins in eight (Fig. 1A; spots 14, 8, 9, 14, and 15) strongly positive anti-3-nitrotyrosine Western blot 2D gel spots were analyzed with LC-MS/MS. The amino acid sequences of four dierent nitrotyrosyl-peptides in ve 2D gel spots (spots 1, 2, 4, 14, and 15) were determined with MS/ MS sequencing (de novo; SEQUEST) and were matched to four dierent proteins. Those four nitrated proteins participate in important physiology process, including neurotransmission, cellular immunity, and cellular structure and mobility; and might provide potential molecular clues to further study the role of NO-induced PTMs in the human pituitary. The human genome contains ca. 30,000 genes that code ca. 100,000 proteins. Most of the proteins distribute on a 2D gel within the area of pH 310. Here, 2DGE was performed with 18-cm IPG strip pH 310 NL. For the pituitary proteome, a 2DGE gel can detect ca. 1000 spots [27,30]. We used LC-MS/MS to analyze the proteins contained within those spots; each spot contained several proteins. In addition to nitrotyrosyl-proteins, high-abundance human growth hormones were also contained in each spot (spots 14, 8, and 9), other types of actin cytoplasmic (SWISS-PROT number P02570 and P02571) in spot 14, and tubulin beta in spot 16, which is consistent with our previous results [30,31]. Other studies also found that one 2D gel spot could include several proteins (six proteins) [32]. The accurate de novo sequencing method, and the less-accurate SEQUEST spectra-comparison method, are two dierent methods to obtain the amino acid sequence of a peptide that had been analyzed in the MS/ MS mode. The basic procedure of SEQUEST analysis is that, for each peptide from a protein in the database, the list of all singly charged ions m/z values and all doubly charged ions m/z values is calculated. The observed m/z values in the product-ion spectrum of a precursor ion are matched and correlated, according to a special statistical model, to those theoretical m/z values. Some indices (e.g., Xcorr, dCn, etc.) are calculated to provide a parameter that represents a goodness-of-t between the theoretical and the measured spectra. The best-t data represent the peptide sequence. For

de novo sequencing, the observed m/z values in the product-ion spectrum of a precursor ion are used to manually deduce the amino acid sequence from the difference between the m/z values of abundant ions. Each amino acid sequence from each method was used to match the protein sequence in a database to determine the protein. For an MS/MS spectrum from a precursor ion, the peptide sequence length derived from SEQUEST analysis might be longer than that sequence from de novo sequencing; for example, for spots 1 and 15 (Table 2). De novo sequencing more accurately determines an amino sequence and the assignment of a Tyr nitration site; especially for a peptide with a nonsignicant SEQUEST Xcorr score, and/or no K/R at the C-terminus, and no K/R preceding the N-terminus. For example, the amino acid residue that precedes the N-terminus of *YTTQNLIR is the acid-labile D residue [29] (Table 2); the puried tryptic peptides were kept 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 because no protease inhibitors were used to extract proteins; for example, some endogenous proteinases may cleave residue I at its C-terminus (http://us.expasy. org/tools/peptidecutter/peptidecutter_enzymes.html). De novo analysis can almost always provide more accurate amino acid sequence than SEQUEST analysis. The integration of Western blotting and LC-MS/MS is an eective approach to detect and characterize nitrotyrosyl-protein in the human pituitary proteome. Twodimensional gel electrophoresis-based Western blotting can pre-separate and enrich proteins with a similar pI and Mr. LC can real-time pre-separate and enrich those tryptic peptides before mass spectrometry analysis. MS/ MS can accurately locate each nitration site. This methodology provides a basis to comprehensively investigate the nitroproteome in the human pituitary, especially to achieve our goal to detect and characterize pituitary adenoma-related nitrotyrosyl-proteins in a program to clarify the basic molecular mechanisms of pituitary adenoma formation.

Acknowledgments The authors gratefully acknowledge nancial assistance (to D.M.D.) from the National Institutes of Health (NS 42843). The MALDI-TOF mass spectrometer was purchased with grants (D.M.D.) from NIH (RR-10522) and NSF (DBI 9604633), and the LCQ with a grant (D.M.D.) from NIH (RR-14593). We acknowledge the helpful discussions with Clive Slaughter. The pituitary control tissues were provided by the Memphis Regional Medical Center (Memphis, TN, USA).

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