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• W2024526046 abstract "A single administration of monocrotaline to rats results in pathologic alterations in the lung and heart similar to human pulmonary hypertension. In order to produce these lesions, monocrotaline is oxidized to monocrotaline pyrrole in the liver followed by hematogenous transport to the lung where it injures pulmonary endothelium. In this study, we determined specific endothelial targets for 14C-monocrotaline pyrrole using two-dimensional gel electrophoresis and autoradiographic detection of protein metabolite adducts. Selective labeling of specific proteins was observed. Labeled proteins were digested with trypsin, and the resulting peptides were analyzed using matrix-assisted laser desorption ionization mass spectrometry. The results were searched against sequence data bases to identify the adducted proteins. Five abundant adducted proteins were identified as galectin-1, protein-disulfide isomerase, probable protein-disulfide isomerase (ER60), β- or γ-cytoplasmic actin, and cytoskeletal tropomyosin (TM30-NM). With the exception of actin, the proteins identified in this study have never been identified as potential targets for pyrroles, and the majority of these proteins have either received no or minimal attention as targets for other electrophilic compounds. The known functions of these proteins are discussed in terms of their potential for explaining the pulmonary toxicity of monocrotaline. A single administration of monocrotaline to rats results in pathologic alterations in the lung and heart similar to human pulmonary hypertension. In order to produce these lesions, monocrotaline is oxidized to monocrotaline pyrrole in the liver followed by hematogenous transport to the lung where it injures pulmonary endothelium. In this study, we determined specific endothelial targets for 14C-monocrotaline pyrrole using two-dimensional gel electrophoresis and autoradiographic detection of protein metabolite adducts. Selective labeling of specific proteins was observed. Labeled proteins were digested with trypsin, and the resulting peptides were analyzed using matrix-assisted laser desorption ionization mass spectrometry. The results were searched against sequence data bases to identify the adducted proteins. Five abundant adducted proteins were identified as galectin-1, protein-disulfide isomerase, probable protein-disulfide isomerase (ER60), β- or γ-cytoplasmic actin, and cytoskeletal tropomyosin (TM30-NM). With the exception of actin, the proteins identified in this study have never been identified as potential targets for pyrroles, and the majority of these proteins have either received no or minimal attention as targets for other electrophilic compounds. The known functions of these proteins are discussed in terms of their potential for explaining the pulmonary toxicity of monocrotaline. monocrotaline pulmonary hypertension monocrotaline pyrrole matrix-assisted laser desorption ionization 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate polyvinylidene difluoride high pressure liquid chromatography adrenocorticotropic hormone post-source decay Tris-buffered saline protein-disulfide isomerase extracellular matrix The pyrrolizidine alkaloid monocrotaline (MCT)1 is a phytotoxin used experimentally to cause a pulmonary vascular syndrome in rats characterized by proliferative pulmonary vasculitis, pulmonary hypertension (PH), and cor pulmonale (1Kay J.M. Heath D. Crotalaria Spectabilis, the Pulmonary Hypertension Plant. C. C. Thomas, Springfield, IL1969: 1-146Google Scholar, 2Wilson D.W. Segall H.J. Pan L.C. Lamé M.W. Estep J.E. Morin D. Crit. Rev. Toxicol. 1992; 22: 307-325Crossref PubMed Scopus (165) Google Scholar, 3Chesney C.F. Allen J.R. Am. J. Pathol. 1973; 70: 489-492PubMed Google Scholar). Although MCT intoxication is used as a model for studying human PH, the initiating mechanism(s) by which this agent produces PH have remained elusive. To produce pulmonary insult, MCT must first be activated by the liver to the putative electrophile monocrotaline pyrrole (MCTP) (4Mattocks A.R. Nature. 1968; 217: 723-728Crossref PubMed Scopus (395) Google Scholar, 5Segall H.J. Wilson D.W. Lamé M.W. Morin D. Winter C.K. Keeler R.F. Tu A.T. Handbook of Natural Toxins, Toxicology of Plant and Fungal Compounds. 6. Marcel Dekker, New York1991: 3-26Google Scholar) which has characteristics of a bifunctional cross-linking agent and has a half-life of ∼3 s in aqueous environments near neutral pH (6Mattocks A.R. Jukes R. Chem. Biol. Interact. 1990; 76: 19-30Crossref PubMed Scopus (37) Google Scholar). Stabilization of MCTP by red blood cells facilitates subsequent transport to the lung (7Pan L.C. Lamé M.W. Morin D. Wilson D.W. Segall H.J. Toxicol. Appl. Pharmacol. 1991; 110: 336-346Crossref PubMed Scopus (38) Google Scholar). The evidence for the involvement of the pulmonary endothelium as the target for MCT intoxication is supported by the circulatory proximity of the liver to the lung endothelium, evidence of increased thymidine uptake and decreased 5-hydroxytryptamine clearance by endothelial cells, and extravasculature leakage of large macromolecules (2Wilson D.W. Segall H.J. Pan L.C. Lamé M.W. Estep J.E. Morin D. Crit. Rev. Toxicol. 1992; 22: 307-325Crossref PubMed Scopus (165) Google Scholar, 8Meyrick B.O. Reid L.M. Am. J. Pathol. 1982; 106: 84-94PubMed Google Scholar, 9Hoorn C.M. Roth R.A. Am. J. Physiol. 1992; 262: L740-L747PubMed Google Scholar). Human primary PH is hypothesized to be an inheritable dysfunction of the pulmonary vascular endothelial cells (10Voelkel N.F. Tuder R.M. Eur. Respir. J. 1995; 8: 2129-2138Crossref PubMed Scopus (174) Google Scholar). In primary PH, disturbance of the endothelial cell surface is suspected to be the initiating factor in the formation of platelet aggregates (11Lopes A.A. Maeda N.Y. Almeida A. Jaeger R. Ebaid M. Chamone D.F. Angiology. 1993; 44: 701-706Crossref PubMed Scopus (30) Google Scholar) and cause the presence of in situ thrombosis (12Fuster V. Steele P.M. Edwards W.D. Gersh B.J. McGoon M.D. Frye R.L. Circulation. 1984; 70: 580-587Crossref PubMed Scopus (915) Google Scholar, 13Wagenvoort C.A. Mulder P.G. Chest. 1993; 103: 844-849Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In vitro experiments with bovine pulmonary artery endothelial cells have shown that MCTP can cause a moderate decrease in their ability to act as a permeability barrier (14Taylor D.W. Wilson D.W. Lamé M.W. Dunston S.D. Jones A.D. Segall H.J. Toxicol. Appl. Pharmacol. 1997; 143: 196-204Crossref PubMed Scopus (25) Google Scholar), cell proliferation is inhibited (15Hoorn C.M. Wagner J.G. Roth R.A. Toxicol. Appl. Pharmacol. 1993; 120: 281-287Crossref PubMed Scopus (21) Google Scholar), prolonged cell cycle arrest in G2-M occurs (16Thomas H.C. Lamé M.W. Wilson D.W. Segall H.J. Toxicol. Appl. Pharmacol. 1996; 141: 319-329Crossref PubMed Google Scholar, 17Thomas H.C. Lamé M.W. Morin D. Wilson D.W. Segall H.J. Am. J. Respir. Cell Mol. Biol. 1998; 19: 129-142Crossref PubMed Scopus (23) Google Scholar), and cells unable to correct or compensate for electrophilic insult often undergo apoptosis (18Thomas H.C. Lamé M.W. Dunston S.K. Segall H.J. Wilson D.W. Toxicol. Appl. Pharmacol. 1998; 151: 236-244Crossref PubMed Scopus (54) Google Scholar). Apoptosis has recently been shown to occur in rat pulmonary artery endothelial cells following the in vivoadministration of MCT (19Jones P.L. Rabinovitch M. Circ. Res. 1996; 79: 1131-1142Crossref PubMed Scopus (157) Google Scholar). Previous work has supported the involvement of endothelial cells as the target for MCT-induced pulmonary hypertension; however, the mechanism(s) by which these cells lose their ability to function correctly is unknown. With respect to pyrrole adduct formation this has been restricted to the measurement of covalent binding to endothelial cell DNA (16Thomas H.C. Lamé M.W. Wilson D.W. Segall H.J. Toxicol. Appl. Pharmacol. 1996; 141: 319-329Crossref PubMed Google Scholar, 20Wagner J.G. Petry T.W. Roth R.A. Am. J. Physiol. 1993; 264: L517-L522PubMed Google Scholar). MCTP has been shown to react in a facile manner with the thiol groups of cysteine and glutathione (21Mattocks A.R. Bird I. Toxicol. Lett. 1983; 16: 1-8Crossref PubMed Scopus (26) Google Scholar, 22Mattocks A.R. Jukes R. Chem. Biol. Interact. 1990; 75: 225-239Crossref PubMed Scopus (43) Google Scholar, 23Reed R.L. Miranda C.L. Kedzierski B. Henderson M.C. Buhler D.R. Xenobiotica. 1992; 22: 1321-1327Crossref PubMed Scopus (40) Google Scholar, 24Lamé M.W. Jones A.D. Morin D. Segall H.J. Wilson D.W. Drug Metab. Dispos. 1995; 23: 422-429PubMed Google Scholar, 25Robertson K.A. Seymour J.L. Hsia M.-T. Allen J.R. Cancer Res. 1977; 37: 3141-3144PubMed Google Scholar). A carbonium ion can be generated at both the C7 and the C9 positions on the pyrrole ring with the pyrrole structure being stabilized by resonance structures that share a charge with the bridge head nitrogen (26Huxtable R.J. Gen. Pharmacol. 1979; 10: 159-167Crossref PubMed Scopus (66) Google Scholar). This delocalization of charge for MCTP confers soft electrophile characteristics (27Pearson R.G. Songstad J. J. Am. Chem. Soc. 1967; 89: 1827-1836Crossref Scopus (938) Google Scholar, 28Coles B. Drug Metab. Rev. 1985; 15: 1307-1334Crossref Scopus (106) Google Scholar) in line more with reactivity toward soft nucleophile protein side chains than with nucleic acids, which are harder nucleophiles. It has previously been shown that MCTP reacts with thiol groups on proteins such as hemoglobin (22Mattocks A.R. Jukes R. Chem. Biol. Interact. 1990; 75: 225-239Crossref PubMed Scopus (43) Google Scholar, 29Seawright A.A. Hrdlicka J. Wright J.D. Kerr D.R. Mattocks A.R. Jukes R. Vet. Hum. Toxicol. 1991; 33: 286-287PubMed Google Scholar, 30Lamé M.W. Jones A.D. Morin D. Wilson D.W. Segall H.J. Chem. Res. Toxicol. 1997; 10: 694-701Crossref PubMed Scopus (16) Google Scholar). Of the limited number of proteins identified as specific targets for MCTP, cytochrome P450 3A, which is responsible for the dehydrogenation of MCT, has also been shown to form adducts with pyrroles (31Reid M.J. Lamé M.W. Morin D. Wilson D.W. Segall H.J. J. Biochem. Mol. Toxicol. 1998; 12: 157-166Crossref PubMed Google Scholar). In this study we have coupled the use of two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) to identify five major MCTP target proteins in human lung endothelial cells and discuss their potential relevance to the enigmatic process of pulmonary hypertension. All reagents unless otherwise indicated were obtained from Fisher. Crotalaria spectabilis was grown under a confined atmosphere of 14CO2, and the14C-monocrotaline (14C-MCT, purity >98%) was extracted and purified as described before (32Lamé M.W. Morin D. Wilson D.W. Segall H.J. J. Label. Compd. Radiopharm. 1996; 38: 1053-1060Crossref Scopus (8) Google Scholar).14C-MCT was converted to 14C-monocrotaline pyrrole (14C-MCTP, 1.95 mCi/mmol) by the method of Mattockset al. (33Mattocks A.R. Jukes R. Brown J. Toxicon. 1989; 27: 561-567Crossref PubMed Scopus (116) Google Scholar) using tetrabromo-1,2-benzoquinone (Aldrich); the product was recrystallized in hexane:diethyl ether. Using fast atom bombardment mass spectrometry the conversion of MCT to MCTP was found to be complete; daughter spectra contained ions characteristic of the pyrrole (34Pan L.C. Wilson D.W. Lamé M.W. Jones A.D. Segall H.J. Toxicol. Appl. Pharmacol. 1993; 118: 87-97Crossref PubMed Scopus (44) Google Scholar). 14C-MCTP was stored inN,N-dimethylformamide at −80 °C until just prior to use. Normal human pulmonary artery endothelial cells (passage 7–8) (Clonetics, San Diego, CA), from a 34-year-old female were grown to 80–90% confluence prior to treatment. Cells in 175-cm2 flasks (Falcon polystyrene) were incubated at 37 °C, 5% CO2 with humidity in EGM-2 medium (Clonetics). The medium was replaced with EGM-2 without 10% fetal bovine serum immediately before exposing cells to 105 μm14C-MCTP delivered in 12.9 μl ofN,N-dimethylformamide/25 ml of media. Cells were removed from flasks using 39-cm cell scrapers (Sarstedt, Newton, NC). After pelleting, the cells were washed three times with isotonic phosphate-buffered saline (136.89 mm NaCl, 2.68 mm KCl, 10.14 mmNa2HPO4, and 1.76 mmKH2PO4, pH, 7.4). The supernatant was removed, and the cells from five flasks were combined, lysed in a 9m urea (ultra pure) solution containing 4% CHAPS (99%), all obtained from Amersham Pharmacia Biotech plus 40 mm Tris and α-toluenesulfonyl fluoride (Aldrich). The latter was added just prior to use in 50 μl of absolute ethanol to give a final concentration of 8 mm. Cells were disrupted with 500 μl of the above, and the lysate was maintained at room temperature for 1 h. The bicinchoninic acid method, as described by the manufacturer (Pierce), was used to determine protein concentrations. Following protein determination, dithiothreitol (Amersham Pharmacia Biotech) was added to give a final concentration of 64.9 mm; lysate was retained at room temperature for an additional 25 min followed by centrifugation (100,000 ×g) for 1 h at 10 °C. Supernatants were stored under N2 at −80 °C until needed. Protein samples (60 μl, 400 μg of protein) were diluted to 250 μl with 8 m urea, 2% CHAPS, 18 mm dithiothreitol, and IPG buffer pH 4–7 was added directly to give a final concentration of 2%. Electrophoresis reagents and hardware, including the Multiphor II platform and EPS 3500XL power supply were purchased from Amersham Pharmacia Biotech. A trace of bromphenol blue was added, and samples (250 μl) were placed in a Immobiline DryStrip reswelling tray. Immobiline DryStrips (IPG) (13 cm, pH 4–7) were placed gel side down in sample, and a layer of mineral oil was added. Samples were allowed to equilibrate with the strips overnight. Isoelectric focusing was performed on a MultiPhor II platform at 15 °C, and strips were covered with mineral oil with the following voltage program: 360 V, 3 h; 1400 V, 0.5 h; 2600 V, 29 h, maximum current 1 mA. IPG strips were stored under N2 in acid-washed glass tubes at −80 °C prior to separation on the second dimension. After removal from storage, strips were allowed to just thaw before being first equilibrated in 30 ml of 50 mm Tris (pH 6.8) containing 30% v/v glycerol (ultra pure), 2% SDS, 6 m urea, 16.2 mmdithiothreitol, and a trace of bromphenol blue, followed by equilibration in the same buffer system (30 ml) with the replacement of dithiothreitol with 135 mm iodoacetamide (97%, Aldrich). Each equilibration phase was of 10-min duration and performed by placing IPG strips in a Petri dish with agitation generated by a rotary shaker. IPG strips along with a 0.5 × 4-mm agarose cylinder containing 0.5 μg of each molecular weight markers (14,000–66,000, Sigma no. MW-SDS-70L) were sealed on top of a 145 × 140 × 1-mm Laemmli (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206908) Google Scholar) separating gel (11% T, 2.7% C, acrylamide (99.9%) and bis-N,N′-methylenebisacrylamide were both obtained from Bio-Rad using 0.5% agarose (SeaKem Gold, FMC, Rockland, ME) dissolved in 125 mm Tris, pH 6.8, 0.1% SDS at 55 °C. Electrophoretic separations were performed at 4 °C, 10 mA/gel. Prior to protein transfers to Sequi-Blot PVDF membranes (0.2 μm, Bio-Rad), gels and membranes were equilibrated for 15 min in transfer buffer (25 mm Tris, 192 mm glycine, 10% methanol). Overnight transfers were conducted using a Transphor (TE 52) tank blotting unit (Hoefer Scientific Instruments, San Francisco, CA), using the above buffer (4 °C), and the electrical conditions were 30 V, 200 mA. Membranes were transferred to glass Pyrex dishes and extensively rinsed with numerous changes of distilled water followed by drying under a stream of N2 and then a final dehydration step in a vacuum desiccator under house vacuum. All glassware used in this process and subsequent digestion protocols were thoroughly cleaned employing sonication in dilute Alconox (Alconox, Inc., New York, NY), and then 0.5 N HCl, distilled water rinses following each procedure coupled with a terminal methanol (HPLC grade) wash. The dried membrane was placed in an autoradiography cassette FBXC and covered with a BioMax TranScreen-LE intensifying screen containing BioMax MS film (Eastman Kodak). Cassettes were stored at −80 °C until the film was developed. After film development, proteins were matched to autoradiographic film spots by staining the membranes for 60 s with 0.005% w/v sulforhodamine B (laser grade, Aldrich) in 30% v/v aqueous methanol containing 0.1% acetic acid with gentle agitation, followed by a 30-s water rinse (36Pappin D.J.C. Smith B.J. Methods in Molecular Biology, Protein Sequencing Protocols. 64. Humana Press, Totowa, NJ1997: 165-173Google Scholar). Proteins containing 14C-pyrrole adducts were excised for tryptic digestion. Sections of membrane containing protein were added to microreaction vessels (0.3 ml) fitted with PTFE-lined caps (Supelco, Bellefonte, PA) and washed with 300 μl of distilled water. Protein spots were then covered with 7 μl of 50 mm ammonium bicarbonate containing 1% n-octyl-β-d-glucopyranoside (Calbiochem) and 280 ng of sequencing grade-modified trypsin (Promega, Madison, WI). Vials were sealed and placed in a secondary container, which was subsequently immersed overnight in a water bath (28 °C). Peptides were extracted from the PVDF matrix by adding 50 μl of a 50% ethanol (Aldrich, spectrophotometric grade): 50% formic acid (purity 99%) to the digestion medium and sonicating vials in a Branson ultrasonic cleaner (Shelton, CT) for 30 min. The extract was concentrated to dryness in 100-μl glass Accuform Micro-Vials (Kimble-Kontes, Vineland, NJ) using a Savant Speed Vac concentrator (Laboratory Equipment Company, Hayward, CA); samples were stored at −80 °C until they were analyzed by MALDI. Molecular masses of tryptic peptides were determined using a Voyager-DE STR MALDI-TOF mass spectrometer (Perseptive Biosystems, Framingham, MA), using a nitrogen laser (337 nm) for ionization. A 10 mg/ml solution of recrystallized α-cyano-4-hydroxycinnamic acid (Aldrich) matrix was prepared in 50% aqueous acetonitrile containing 0.3% trifluoroacetic acid. Peptide digests were dissolved in 10 μl of 50% aqueous acetonitrile containing 0.1% formic acid, and a 0.5-μl aliquot was mixed on the sample target with 0.5 μl of matrix solution and allowed to dry under ambient conditions. Multipoint mass axis calibration was performed using external standards angiotensin I, ACTH(1–17), ACTH(18–39), ACTH(7–38), and bovine insulin. Initial screening was performed in linear mode, and more accurate monoisotopic peptide masses were determined in reflector mode. To determine the identity of selected spots, MS-Fit and in some cases MS-Tag were used to search data bases for peptide mass fingerprints and to match fragment ions observed in post-source decay (PSD) spectra, respectively (37Qiu Y. Benet L.Z. Burlingame A.L. J. Biol. Chem. 1998; 273: 17940-17953Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 38Clauser, K. R., Baker, P., and Burlingame, A. L. (1996) in Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics Portland, OR, May 12–16, p. 365Google Scholar). In addition to the above data bases we have used the ExPASy Proteomics tool ProtScale to accomplish hydropathicity calculations (39Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17157) Google Scholar). These calculations were used to determine the potential accessibility of cysteine residues to MCTP. An additional blot and the corresponding autoradiographic analysis were performed for antibody detection of PDI and galectin-1. PVDF membranes were stained with sulforhodamine B, and the proteins were matched with autoradiographic spots. The location of spots previously identified by peptide mass fingerprinting to correspond to galectin-1 and PDI were marked by recording their horizontal and vertical position prior to destaining with 100% methanol and a final rinse with 70% acetonitrile. Blots were sectioned in half, and one portion developed for galectin-1 and the other PDI. Membranes were first blocked with 3% nonfat milk (Bio-Rad) in 150 mm NaCl, 50 mmTris buffer (TBS), pH 7.4, for 30 min at ambient conditions. This was followed by an overnight incubation with primary antibodies at 4 °C. The primary antibodies were polyclonal rabbit-raised against rat galectin-1 (generously provided by D. N. W. Cooper from UCSF), used at a concentration of 100 μl of serum/20 ml of 3% milk TBS and a mouse monoclonal raised against rat protein-disulfide isomerase synthetic peptide (amino acids 499–509) with a working concentration of 2 μg/ml. The latter was obtained from StressGen Biotechnologies Corp., Victoria, British Columbia, Canada and is known to cross-react with human PDI and calreticulin. After the overnight incubation the blots were washed four times with TBS, and the secondary antibody either goat anti-rabbit or anti-mouse conjugated with alkaline phosphatase (Bio-Rad) was applied at a 1/2000 dilution in 3% milk TBS. Incubations were carried out for 1.5 h at ambient conditions, and the blots were subsequently washed with 0.05% Tween-20 in TBS (two times) followed by four washes with TBS. Blots were developed for 10 min with alkaline phosphatase conjugation substrate kit (Bio-Rad). Results recorded in Fig.1 show that a selective number of proteins form covalent adducts with MCTP. This pattern was consistent from separation to separation, with six separate autoradiographic profiles showing the identical pattern of labeling. Of the 13 labeled spots, seven were chosen for analysis based on the amount of radioactivity associated with them and the intensity of the corresponding sulforhodamine B stain. The latter was used to pick protein spots that were in sufficient quantity and purity to have a reasonable chance of producing an unambiguous match with protein data bases. Of these seven spots, five were identified (TableI) as probable protein-disulfide isomerase precursor ER-60 (EC 5.3.4.1, Swiss-Prot P30101), protein-disulfide isomerase precursor (PDI, EC 5.3.4.1, Swiss-ProtP07237), β- or γ-cytoplasmic actin (Swiss-Prot P02570 and P02571, respectively), cytoskeletal tropomyosin (TM30-NM, Swiss-Prot P12324), and galectin-1 (Swiss-Prot P09382). As recorded in Table I, both the apparent molecular weights and estimated pIs from the two-dimensional separations were in close agreement to data base values with the actual tryptic peptide masses showing excellent coverage and agreement with the expected theoretical values. Typical MALDI mass spectra for tryptic digests of galectin-1 (linear mode) and PDI (reflector mode) are shown in Figs. 2 and3, respectively. PSD spectra were generated to confirm assignments of tryptic fragments from each of these two proteins. The PSD ions described here follow the nomenclature of Biemann (40Biemann K. Methods Enzymol. 1990; 193: 886-888Crossref PubMed Scopus (423) Google Scholar); fragmentation designation is followed in parenthesis by the m/z value. For galectin-1 the peptide sequence DSNNLCLHFNPR was chosen. The PSD ions showed the expected amino acid immonium ions and numerous other expected fragments. Some of the more prominent ions were the C-terminal ions y2 (272), y2-NH3 (255), and y11 (1372); N-terminal ions b4 (431), b6-H2O (686), and c5 (561); and internal fragment ions NLCL (b ion, 501) and CLHFN (b ion, 672). For PDI the peptide ILFIFIDSDHTDNQR was subjected to PSD. Fragmentation for this peptide was not as informative as that observed for the galectin-1 peptide because of the complexity of the PSD spectrum, but the immonium ions for histidine, isoleucine/leucine, and phenylalanine were present. Some of the more prominent ions included m/z 1790, which represented the MH+-45 (loss of the threonine side chain), y3(417), NQ-NH3 (b ion, 226), FIFIDSDHT-H2O (b ion, 1059), LFIFIDSDHT (b ion, 1190), and FIFIDSDHTD (a ion, 1164). Spots three and five produced no meaningful matches considering apparent molecular weight, estimated pI, or the peptide mass fingerprint. There are also a number of discrete radiolabeled spots that travel with the dye front. Because the number of known metabolites and break down products of MCTP (24Lamé M.W. Jones A.D. Morin D. Segall H.J. Wilson D.W. Drug Metab. Dispos. 1995; 23: 422-429PubMed Google Scholar, 41Lamé M.W. Jones A.D. Morin D. Segall H.J. Drug Metab. Dispos. 1991; 19: 516-524PubMed Google Scholar, 42Lamé M.W. Morin D. Jones A.D. Segall H.J. Wilson D.W. Toxicol. Lett. 1990; 51: 321-329Crossref PubMed Scopus (52) Google Scholar) cannot account for this heavily labeled area, we suspect that these are proteolysis products derived from adducted proteins.Table ISummary of MALDI masses obtained from tryptic digests of pyrrole adducted proteinsSpot no.ExperimentalΔ1-aThe difference between the measured monoisotopic mass and calculated mass.Proposed peptide sequence1-bParentheses denote residue before and after the peptide. Cim, denotes iodoacetamide-derivatized Cys; Ac-, acetylated N terminus; Met-ox, methionine sulfoxide; pyro-Glu, N-terminal Gln converted to pyroglutamine.Peptide sequence numbersProtein match (Swiss-Prot accession no.)Percentage of sequence coverage observed in MALDI spectrumCalculated molecular mass1-cMasses according to MS-FIT.Molecular mass from gelCalculated pIpI from gelm/zmDaDakDa1608.299−41.4(K)LSNFK(T)272–276Protein-disulfide isomerase (precursor), human (P07237)43%57,11658,9004.764.8761.341−38.6(K)AEGSEIR(L)72–78763.385−50.8(K)IFGGEIK(T)248–254777.420−31.1(K)DGVVLFK(K)201–207870.346−38.8(K)SVSDYDGK(L)264–271910.416−25.9(K)FFPASADR(T)445–452928.484−41.7(K)VHSFPTLK(F)437–444962.411−41.2(R)ITEFCimHR(F)339–345966.528−38.2(R)ILEFFGLK(K)301–308988.4912.6(K)KEECimPAVR(L)309–316991.524−22.6(K)ENLLDFIK(H)223–2301002.471−87.4(K)LKAEGSEIR(L)70–781037.424−66.4(R)NNFEGEVTK(E)214–2221066.470−46.9(R)TVIDYNGER(T)453–4611081.635−42.4(K)THILLFLPK(S)255–2631158.539−51.7(K)SNFAEALAAHK(Y)32–421213.501−36.7(K)NFEDVAFDEK(K)376–3851451.680−21.8(K)YKPESEELTAER(I)327–3381780.794−41.3(K)VDATEESDLAQQYG- VR(G)82–971833.884−29.5(K)ILFIFIDSDHTDNQ- R(I)286–3001965.005−39.6(K)HNQLPLVIEFTEQTA- PK(I)231–2472935.451−43.1(R)TGPAATTLPDGAAA- ESLVESSEVAVIGFFK- (D)133–1622877.4999.7(K)LNFAVASR(K)297–304Probable protein-disulfide isomerase, human (P30101), ER6031%56,78259,8005.9861172.56827(K)FVMQEEFSR(D)336–3441188.55519.1(K)FVMet-oxQEEFSR(D)336–3441191.582−19(R)LAPEYEAAATR(L)63–731236.53925.7(R)DGEEAGAYDGPR(T)108–1191341.70419.7(R)GFPTIYFSPANK(K)449–4601359.67415.5(R)FLQDYFDGNLK(R)352–3621368.72156.3(K)SEPIPESNDGPVK(V)367–3791370.72630.4(R)ELSDFISYLQR(E)472–4821619.736−48.5(K)DLLIAYYDVDYEK- (N)259–2711664.750−9(K)MDATANDVPSPYEV- R(G)434–4481680.81056.1(K)Met-oxDATA- NDVPSPYEVR(G)434–4482348.263190.7(K)DASIVGFFDDSFS- EAHSEFLK(A)153–1732575.33833.6(K)TFSHELSDFGLES- TAGEIPVVAIR(T)306–3294644.352−21(R)LDLAGR(D)178–183β-actin (P02570) or γ-actin (P02571) cytoplasmic, human29%41,73747,7005.295.4795.459−13.9(K)IIAPPER(K)329–335417935.31976.4491.1(K)AGFAGDDAPR(A)19–281132.5324.5(R)GYSFTTTAER(E)197–2061499.69114.3(K)pyro-GluEYDESGPSIVHR(K)360–3721515.76919.3(K)IWHHTFYNELR(V)85–951516.73026.8(K)QEYDESGPSIVH- R(K)360–3721790.92633.5(K)SYELPDGQVITI- GNER(F)239–2541954.09732(R)VAPEEHPVLLTE- APLNPK(A)96–1132231.057−8.4(K)DLYANTVLSGGTT- Met-oxYPGIADR- (M)292–3126630.38123.7(K)VIENR(A)93–97Tropomyosin, cytoskeletal, human (P12324)26%29,03235,6004.754.9718.37336.2(R)EVEGER(R)34–39718.37311.1(K)LEEAEK(A)77–82722.37629(R)AEFAER(G)203–208777.37840.1(K)AADESER(G)83–89894.52657.9(R)KYEEVAR(K)125–131894.52657.9(K)YEEVARK(L)126–132940.50354.8(K)HIAEEADR(K)117–1241156.74587.2(K)LVIIEGDLER(T)133–1421243.75298.6(R)IQLVEEELDR(A)56–651284.84491.3(R)KLVIIEGDLER(T)132–1427786.399−0.7(R)GEVAPDAK(S)21–28Galectin-1, human (P09382)74%14,58414,1005.345.1968.49118.1(K)LPDGYEFK(F)100–1071041.60535.7(R)VRGEVAPDAK(S)19–281076.50124.9(K)DGGAWGTEQR(E)64–731486.73447.9(K)DSNNLCimLHFN- PR(F)37–481800.89749.2(R)LNLEAINYMet-oxAADGDFK(I)112–1272014.06947.6(−)Ac-ACimGLV- ASNLNLKPGECim- LR(V)1-dAcetylated alanine labeled as first residue.1–182867.49061(R)EAVFPFQPGSVAE- VCimITFDQANLTVK(L)74–991-a The difference between the measured monoisotopic mass and calculated mass.1-b Parentheses denote residue before and after the peptide. Cim, denotes iodoacetamide-derivatized Cys; Ac-, acetylated N terminus; Met-ox, methionine sulfoxide; pyro-Glu, N-terminal Gln converted to pyroglutamine.1-c Masses according to MS-FIT.1-d Acetylated alanine labeled as first residue. Open table in a new tab Figure 2MALDI spectrum (linear mode) of peptides derived from the digestion of galectin-1 with trypsin. Numbers positioned above ions represent the peptide sequence in the parent protein. Ion masses corresponding to the tryptic fragments are listed in Table I, except for span 29–36 (m/z 878) and the peptide generated by a single missed cleavage by trypsin 74–111 (m/z 4334).View Large Image Figure ViewerDownload (PPT)Figure 3MALDI spectrum (reflector mode) of peptides derived from the digestion of PDI with trypsin. Numbers positioned above ions represent the peptide sequence in the parent protein. Ion masses corresponding to the respective trypsin-generated peptides are listed in Table I.View Large Image Figure ViewerDownload (PPT) Because these are two of the more interesting proteins found to be adducted by pyrroles, we determined if for future experiments they could be more simply identified through the use of antibody techniques. Results are recorded in Fig. 4. The commercially av" @default.
• W2024526046 created "2016-06-24" @default.
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• W2024526046 date "2000-09-01" @default.
• W2024526046 modified "2023-09-26" @default.
• W2024526046 title "Protein Targets of Monocrotaline Pyrrole in Pulmonary Artery Endothelial Cells" @default.
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• W2024526046 doi "https://doi.org/10.1074/jbc.m001372200" @default.
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