Artigo Acesso aberto Revisado por pares

A Filtered Database Search Algorithm for Endogenous Serum Protein Carbonyl Modifications in a Mouse Model of Inflammation

2011; Elsevier BV; Volume: 10; Issue: 10 Linguagem: Inglês

10.1074/mcp.m111.007658

ISSN

1535-9484

Autores

Peter G. Slade, Michelle V. Williams, Alison Chiang, Elizabeth Iffrig, Steven R. Tannenbaum, John S. Wishnok,

Tópico(s)

Adipose Tissue and Metabolism

Resumo

During inflammation, the resulting oxidative stress can damage surrounding host tissue, forming protein-carbonyls. The SJL mouse is an experimental animal model used to assess in vivo toxicological responses to reactive oxygen and nitrogen species from inflammation. The goals of this study were to identify the major serum proteins modified with a carbonyl functionality and to identify the types of carbonyl adducts. To select for carbonyl-modified proteins, serum proteins were reacted with an aldehyde reactive probe that biotinylated the carbonyl modification. Modified proteins were enriched by avidin affinity and identified by two-dimensional liquid chromatography tandem MS. To identify the carbonyl modification, tryptic peptides from serum proteins were subjected to avidin affinity and the enriched modified peptides were analyzed by liquid chromatography tandem MS. It was noted that the aldehyde reactive probe tag created tag-specific fragment ions and neutral losses, and these extra features in the mass spectra inhibited identification of the modified peptides by database searching. To enhance the identification of carbonyl-modified peptides, a program was written that used the tag-specific fragment ions as a fingerprint (in silico filter program) and filtered the mass spectrometry data to highlight only modified peptides. A de novo-like database search algorithm was written (biotin peptide identification program) to identify the carbonyl-modified peptides. Although written specifically for our experiments, this software can be adapted to other modification and enrichment systems. Using these routines, a number of lipid peroxidation-derived protein carbonyls and direct side-chain oxidation proteins carbonyls were identified in SJL mouse serum. During inflammation, the resulting oxidative stress can damage surrounding host tissue, forming protein-carbonyls. The SJL mouse is an experimental animal model used to assess in vivo toxicological responses to reactive oxygen and nitrogen species from inflammation. The goals of this study were to identify the major serum proteins modified with a carbonyl functionality and to identify the types of carbonyl adducts. To select for carbonyl-modified proteins, serum proteins were reacted with an aldehyde reactive probe that biotinylated the carbonyl modification. Modified proteins were enriched by avidin affinity and identified by two-dimensional liquid chromatography tandem MS. To identify the carbonyl modification, tryptic peptides from serum proteins were subjected to avidin affinity and the enriched modified peptides were analyzed by liquid chromatography tandem MS. It was noted that the aldehyde reactive probe tag created tag-specific fragment ions and neutral losses, and these extra features in the mass spectra inhibited identification of the modified peptides by database searching. To enhance the identification of carbonyl-modified peptides, a program was written that used the tag-specific fragment ions as a fingerprint (in silico filter program) and filtered the mass spectrometry data to highlight only modified peptides. A de novo-like database search algorithm was written (biotin peptide identification program) to identify the carbonyl-modified peptides. Although written specifically for our experiments, this software can be adapted to other modification and enrichment systems. Using these routines, a number of lipid peroxidation-derived protein carbonyls and direct side-chain oxidation proteins carbonyls were identified in SJL mouse serum. During inflammation, activated phagocytes secrete reactive nitrogen species (RNS) and reactive oxygen species (ROS) that can eliminate infectious agents. If inflammation is chronic, RNS and ROS can also damage surrounding host tissue, resulting in protein modification in the form of protein-carbonyls (1Grimsrud P.A. Xie H. Griffin T.J. Bernlohr D.A. Oxidative stress and covalent modification of protein with bioactive aldehydes.J. Biol. Chem. 2008; 283: 21837-21841Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Total protein carbonylation has been used as a marker of oxidative stress and inflammation and increased levels have been seen in heart disease, lung disease, aging, neurodegenerative disorders, and inflammatory bowel disease (2Dalle-Donne I. Giustarini D. Colombo R. Rossi R. Milzani A. Protein carbonylation in human diseases.Trends Mol. Med. 2003; 9: 169-176Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar, 3Butterfield D.A. Kanski J. Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins.Mech. Ageing Dev. 2001; 122: 945-962Crossref PubMed Scopus (361) Google Scholar, 4Buss I.H. Darlow B.A. Winterbourn C.C. Elevated protein carbonyls and lipid peroxidation products correlating with myeloperoxidase in tracheal aspirates from premature infants.Pediatr. Res. 2000; 47: 640-645Crossref PubMed Scopus (80) Google Scholar, 5Blackburn A.C. Doe W.F. Buffinton G.D. Protein carbonyl formation on mucosal proteins in vitro and in dextran sulfate-induced colitis.Free Radic. Biol. Med. 1999; 27: 262-270Crossref PubMed Scopus (42) Google Scholar, 6Chaudhuri A.R. de Waal E.M. Pierce A. Van Remmen H. Ward W.F. Richardson A. Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteomic approach.Mech. Ageing Dev. 2006; 127: 849-861Crossref PubMed Scopus (130) Google Scholar, 7Serdar Z. Serdar A. Altin A. Eryilmaz U. Albayrak S. The relation between oxidant and antioxidant parameters and severity of acute coronary syndromes.Acta Cardiol. 2007; 62: 373-380Crossref PubMed Scopus (30) Google Scholar). The carbonylation of proteins can result from the direct oxidation of protein side-chains, forming the glutamate and aminoadipate semialdehydes (Scheme 1) (8Requena J.R. Chao C.C. Levine R.L. Stadtman E.R. Glutamic and aminoadipic semialdehydes are the main carbonyl products of metal-catalyzed oxidation of proteins.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 69-74Crossref PubMed Scopus (374) Google Scholar, 9Yuan Q. Zhu X. Sayre L.M. Chemical nature of stochastic generation of protein-based carbonyls: metal-catalyzed oxidation versus modification by products of lipid oxidation.Chem. Res. Toxicol. 2007; 20: 129-139Crossref PubMed Scopus (56) Google Scholar), but can also occur from the indirect oxidation of polyunsaturated fatty acids (lipid peroxidation) and carbohydrates, leading to a variety of reactive aldehydes (Scheme 2) (10Sayre L.M. Lin D. Yuan Q. Zhu X. Tang X. Protein adducts generated from products of lipid oxidation: focus on HNE and ONE.Drug Metab. Rev. 2006; 38: 651-675Crossref PubMed Scopus (290) Google Scholar). These aldehydes covalently modify proteins through conjugate addition (often Michael addition) to nucleophilic amino acid side chains, creating protein-bound carbonyls (10Sayre L.M. Lin D. Yuan Q. Zhu X. Tang X. Protein adducts generated from products of lipid oxidation: focus on HNE and ONE.Drug Metab. Rev. 2006; 38: 651-675Crossref PubMed Scopus (290) Google Scholar, 11Refsgaard H.H. Tsai L. Stadtman E.R. Modifications of proteins by polyunsaturated fatty acid peroxidation products.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 611-616Crossref PubMed Scopus (281) Google Scholar).Scheme 2Reactive aldehydes, arising from oxidation of polyunsaturated fatty acids and carbohydrates, can indirectly lead to protein carbonylation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In a previous study, DNA oxidative damage products, from tissues from the SJL mouse model of inflammation, were quantitated (12Pang B. Zhou X. Yu H. Dong M. Taghizadeh K. Wishnok J.S. Tannenbaum S.R. Dedon P.C. Lipid peroxidation dominates the chemistry of DNA adduct formation in a mouse model of inflammation.Carcinogenesis. 2007; 28: 1807-1813Crossref PubMed Scopus (102) Google Scholar). Only the lipid peroxidation adducts increased in association with inflammation, which suggested an important role of lipids in inflammatory disease progression and established a direct correlation between inflammation and the increased formation of reactive aldehydes from oxidized lipids. Although DNA modification because of inflammation has been the focus of many animal and human studies, it is proteins that are considered most likely to be ubiquitously affected by disease, response, and recovery (13Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility.Nat. Biotechnol. 2006; 24: 971-983Crossref PubMed Scopus (1371) Google Scholar), and the biological consequences include more rapid protein turnover as well as novel signaling (14Rouzer C.A. Marnett L.J. Structural and functional differences between cyclooxygenases: fatty acid oxygenases with a critical role in cell signaling.Biochem. Biophys. Res. Commun. 2005; 338: 34-44Crossref PubMed Scopus (39) Google Scholar, 15West J.D. Marnett L.J. Alterations in gene expression induced by the lipid peroxidation product, 4-hydroxy-2-nonenal.Chem Res. Toxicol. 2005; 18: 1642-1653Crossref PubMed Scopus (110) Google Scholar, 16Marnett L.J. Riggins J.N. West J.D. Endogenous generation of reactive oxidants and electrophiles and their reactions with DNA and protein.J. Clin. Invest. 2003; 111: 583-593Crossref PubMed Scopus (397) Google Scholar). The overall carbonylation of proteins has been well documented in other inflammatory animal models, which have shown significant increases in protein-carbonyls in the mucosal lining of rat colon (17Pélissier M.A. Marteau P. Pochart P. Antioxidant effects of metronidazole in colonic tissue.Dig. Dis. Sci. 2007; 52: 40-44Crossref PubMed Scopus (15) Google Scholar) and mouse colon (5Blackburn A.C. Doe W.F. Buffinton G.D. Protein carbonyl formation on mucosal proteins in vitro and in dextran sulfate-induced colitis.Free Radic. Biol. Med. 1999; 27: 262-270Crossref PubMed Scopus (42) Google Scholar) whereas increased levels of protein carbonyls were observed in rat serum, along with a higher turnover of proteins from the inflamed tissue (18Mercier S. Breuillé D. Mosoni L. Obled C. Patureau M.P. Chronic inflammation alters protein metabolism in several organs of adult rats.J. Nutr. 2002; 132: 1921-1928Crossref PubMed Scopus (70) Google Scholar, 19El Yousfi M. Breuillé D. Papet I. Blum S. André M. Mosoni L. Denis P. Buffière C. Obled C. Increased tissue protein synthesis during spontaneous inflammatory bowel disease in HLA-B27 rats.Clin. Sci. 2003; 105: 437-446Crossref Scopus (24) Google Scholar). Furthermore, increased protein carbonyl modification has been reported in studies of the colon mucosal lining from patients diagnosed with inflammatory bowel disease (20Keshavarzian A. Banan A. Farhadi A. Komanduri S. Mutlu E. Zhang Y. Fields J.Z. Increases in free radicals and cytoskeletal protein oxidation and nitration in the colon of patients with inflammatory bowel disease.Gut. 2003; 52: 720-728Crossref PubMed Scopus (189) Google Scholar, 21Lih-Brody L. Powell S.R. Collier K.P. Reddy G.M. Cerchia R. Kahn E. Weissman G.S. Katz S. Floyd R.A. McKinley M.J. Fisher S.E. Mullin G.E. Increased oxidative stress and decreased antioxidant defenses in mucosa of inflammatory bowel disease.Dig. Dis. Sci. 1996; 41: 2078-2086Crossref PubMed Scopus (427) Google Scholar). Taken together, these observations suggest that an increase in carbonylated proteins is likely in the SJL mouse and that the extent and type of protein-carbonyls could potentially be a marker for inflammation and disease. The SJL mouse is an experimental animal model used to assess in vivo toxicological responses to nitric oxide (NO) overproduction from inflammation (22Gal A. Tamir S. Tannenbaum S.R. Wogan G.N. Nitric oxide production in SJL mice bearing the RcsX lymphoma: A model for in vivo toxicological evaluation of NO.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 11499-11503Crossref PubMed Scopus (31) Google Scholar). Injections of RscX lymphoma cells into these mice result in rapid tumor growth as well as host T-cell proliferation in lymph nodes, spleen, and liver, resulting in morbidity within 15 days. The induced macrophages create a 50-fold increase in NO production in spleen and lymph nodes and the post-translational modification 3-nitrotyrosine was highly elevated in spleen tissue. The identification of endogenously formed protein carbonyls in serum is challenging because of their low abundance and the large number of possible modifications (1Grimsrud P.A. Xie H. Griffin T.J. Bernlohr D.A. Oxidative stress and covalent modification of protein with bioactive aldehydes.J. Biol. Chem. 2008; 283: 21837-21841Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 2Dalle-Donne I. Giustarini D. Colombo R. Rossi R. Milzani A. Protein carbonylation in human diseases.Trends Mol. Med. 2003; 9: 169-176Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar, 23Han B. Stevens J.F. Maier C.S. Design, synthesis, and application of a hydrazide-functionalized isotope-coded affinity tag for the quantification of oxylipid-protein conjugates.Anal. Chem. 2007; 79: 3342-3354Crossref PubMed Scopus (60) Google Scholar), some of which are shown in Schemes 1 and 2. We recently identified proteins modified by the carbonyl 9,12-dioxo-10(E)-dodecenoic acid (DODE) in cells treated with the hydroperoxide of linoleic acid (13-HPODE) (24Slade P.G. Williams M.V. Brahmbhatt V. Dash A. Wishnok J.S. Tannenbaum S.R. Proteins modified by the lipid peroxidation aldehyde 9,12-dioxo-10(E)-dodecenoic acid in MCF7 breast cancer cells.Chem. Res. Toxicol. 2010; 23: 557-567Crossref PubMed Scopus (14) Google Scholar). This work used a technique first demonstrated by Maier and coworkers (25Chavez J. Wu J. Han B. Chung W.G. Maier C.S. New role for an old probe: affinity labeling of oxylipid protein conjugates by N′-aminooxymethylcarbonylhydrazino d-biotin.Anal. Chem. 2006; 78: 6847-6854Crossref PubMed Scopus (71) Google Scholar, 26Chung W.G. Miranda C.L. Maier C.S. Detection of carbonyl-modified proteins in interfibrillar rat mitochondria using N′-aminooxymethylcarbonylhydrazino-D-biotin as an aldehyde/keto-reactive probe in combination with Western blot analysis and tandem mass spectrometry.Electrophoresis. 2008; 29: 1317-1324Crossref PubMed Scopus (42) Google Scholar). Protein carbonyls were derivatized with an aldehyde reactive probe (ARP), 1The abbreviations used are:ARPaldehyde reaction probeBPIbiotin peptide identificationDODE9,12-dioxo-10(E)-dodecenoic acid2D-LC-MS/MStwo-dimensional liquid chromatography tandem MSHNE4-hydroxy-2(E)-nonenalONE4-oxo-2(E)-nonenalSCXstrong cation exchangePBSphosphate-buffered saline. a biotinylated hydroxylamine that reacts preferentially with aldehyde and keto groups (27Ide H. Akamatsu K. Kimura Y. Michiue K. Makino K. Asaeda A. Takamori Y. Kubo K. Synthesis and damage specificity of a novel probe for the detection of abasic sites in DNA.Biochemistry. 1993; 32: 8276-8283Crossref PubMed Scopus (119) Google Scholar), allowing for subsequent enrichment of the modified proteins by avidin affinity. DODE-modified proteins were also identified using an anti-DODE antibody and Western blots. Although a number of DODE modified proteins were identified, we were unable to definitively identify the carbonyl modified peptides by mass spectrometry due both to low abundance and to the interference of ARP-tag-specific fragment ions on database searching. aldehyde reaction probe biotin peptide identification 9,12-dioxo-10(E)-dodecenoic acid two-dimensional liquid chromatography tandem MS 4-hydroxy-2(E)-nonenal 4-oxo-2(E)-nonenal strong cation exchange phosphate-buffered saline. In this current study, SJL mouse serum was screened for the presence of protein carbonyls endogenously formed during inflammation. Carbonyl-modified proteins were then identified using techniques previously established (24Slade P.G. Williams M.V. Brahmbhatt V. Dash A. Wishnok J.S. Tannenbaum S.R. Proteins modified by the lipid peroxidation aldehyde 9,12-dioxo-10(E)-dodecenoic acid in MCF7 breast cancer cells.Chem. Res. Toxicol. 2010; 23: 557-567Crossref PubMed Scopus (14) Google Scholar); first anti-DODE Western blotting followed by ARP derivatization/enrichment and two-dimensional liquid chromatography tandem MS (2D-LC-MS/MS). These proteins then formed a database of putative carbonyl-modified proteins from SJL mouse serum. To identify the type of carbonyl modification and the modified peptide, the ARP derivatized peptides were enriched and analyzed by mass spectrometry. To minimize the confounding effect of ARP fragmentation, an algorithm (in silico filter) was written that filtered the mass spectrometry data to select only those peptides containing the known ARP pattern of fragmentation. This alone effectively reduced the number of false positives. To further alleviate the interfering effects of ARP fragments on peptide identification by database searching, a de novo searching algorithm (Biotin Peptide Identification program, BPI) was written. Peptides were evaluated against the database of proteins that had been previously identified as potentially carbonyl modified. Because modified peptides were searched against a finite list of proteins and all results were manually evaluated, the BPI program did not calculate a statistical peptide score, which allowed the identification of lower abundant modified peptides that would not be considered significant by standard search engines such as Mascot. The BPI program was also written with the flexibility to evaluate a wide range of known carbonyl-adduct masses and could therefore screen for a large number of carbonyl adducts at one time. This should also allow the program to be used with modification/enrichment systems other than the one used here. The program thus selected a finite number of carbonyl modified peptides, resulting in the identification of a number of proteins that were endogenously carbonylated in serum from the SJL mouse inflammation model. Aldehyde reactive probe (ARP) was purchased from Invitrogen (Eugene, OR) and biotin-PEO-LC-Amine was purchased from Pierce (Rockford, IL). Cytochrome c (equine heart), acetic acid, and trifluoroacetic acid were purchased from Sigma Chemical Co. (St. Louis, MO). Trypsin was purchased from Promega (Madison, WI). Gases were supplied by AirGas (Salem, NH). DODE was a generous gift from Prof. Ian A. Blair (University of Pennsylvania). RcsX cells (kindly supplied by Prof. Nicolas M. Ponzio, University of New Jersey Medical Center, Newark, NJ) were passaged through SJL mice (Jackson Laboratory, Bar Harbor, ME) and harvested from lymph nodes 14 days after inoculation according to published procedures (28Gal A. Tamir S. Kennedy L.J. Tannenbaum S.R. Wogan G.N. Nitrotyrosine formation, apoptosis and oxidative damage: relationships to nitric oxide production in SJL mice bearing the RcsX tumor.Cancer Res. 1997; 57: 1823-1828PubMed Google Scholar). Cells were manually dissociated from lymph nodes followed by washing in phosphate-buffered saline (PBS; 140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, pH 7.4) and freezing in aliquots of 5 × 107 cells in 10% dimethyl sulfoxide/fetal bovine serum. To initiate NO overproduction, eight SJL mice (5–6 weeks old) were each injected intraperitoneally with 107 RcsX cells in 200 μl of PBS. Ten mice were injected with 200 μl of PBS as unstimulated controls. Twelve days after injection, five treated and three control mice were anesthetized with isofluorane, and serum was collected by cardiac puncture. Pooled samples were desalted by filter centrifugation and dried in a SpeedVac. Protein content was determined with a commercial bicinchoninic acid (BCA) protein assay kit (Pierce). One hundred micrograms (Coomassie) or 30 μg (Western) of sample was processed by two-dimensional gel electrophoresis for protein identification and Western blotting. For anti-biotin Western blotting and Coomassie, proteins were focused on 11 cm 4–7 immobilized pH gradient (IPG) strips (Immobiline™ DryStrip gels, Amersham Biosciences) using an IPGphor focusing apparatus (Amersham Biosciences). For anti-DODE Western blotting and Coomassie staining, proteins were focused on an 11 cm 3–10 pH gradient strip. Samples were applied by cup loading. IPG strips were then equilibrated in equilibration buffer (50 mm Tris-HCl, 6 m urea, 30% glycerol, 2% SDS) supplemented with 1% dithiothreitol to maintain the fully reduced state of proteins, followed by 2.5% iodoacetamide to prevent re-oxidation of thiol groups during electrophoresis. Proteins were separated on 12.5% Tris/Glycine gels (BioRad) using a Criterion System (BioRad). Proteins were visualized by Coomassie SimplyBlue SafeStain™ (Invitrogen). Proteins were transferred to a polyvinylidine fluoride membrane (BioRad). Precision Plus protein standard (BioRad) was used to estimate molecular weights. Anti-biotin (Cell Signaling, Danvers, MA) 1:20,000 was used to detect biotinylated proteins. Polyclonal anti-DODE antibody (24Slade P.G. Williams M.V. Brahmbhatt V. Dash A. Wishnok J.S. Tannenbaum S.R. Proteins modified by the lipid peroxidation aldehyde 9,12-dioxo-10(E)-dodecenoic acid in MCF7 breast cancer cells.Chem. Res. Toxicol. 2010; 23: 557-567Crossref PubMed Scopus (14) Google Scholar) 1:1500 was used to detect DODE-modified proteins. Protein spots were picked and washed in Milli-Q® water for 15 min, then washed three times in 25 mm NH4HCO3/50% CH3CN for 30 min. Gel plugs were dehydrated in 100% CH3CN for 10 min while vortex-mixing. The supernatant was removed, and the plugs were dried in the SpeedVac. Trypsin (1 μg/50 μl) (Promega, Madison, WI) suspended in 25 mm NH4HCO3 was added, and gel plugs were rehydrated for 30 min on ice and then digested overnight at 37 °C. The samples were then centrifuged, and the supernatant was removed. The pellet was resuspended in CH3CN with 1% TFA, vortexed, and sonicated for 30 min to release hydrophobic peptides. The supernatant was removed and combined with the previous supernatant and stored at −20 °C until ready for MS/MS analysis. Neutravidin was used to enrich for ARP-carbonyl derivatized proteins from SJL serum. These proteins were identified (described below) to make up the protein database used in the Biotin Peptide Identification program. Immobilized neutravidin (Pierce, Rockford, IL) was packed into a column with dimensions ID = 9 mm, OD = 7 mm, height = 60 mm. The final column volume was 2 ml. All buffers and samples were brought to room temperature. The column was washed with 4 column volumes of PBS. A protease inhibitor mixture was added to the protein samples: AEBSF (1 mm), E-64 (10 μm), pepstatin A (1.4 μm), EDTA (1 mm), bestatin (40 μm). Samples (∼2 mg protein in 1 ml PBS) were added directly to the neutravidin column. To remove nonbiotinylated proteins, the column was washed with 10 column volumes of PBS-2% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (CHAPS), five column volumes of PBS and five column volumes of deionized water. Wash fractions were analyzed by UV-Vis spectrometry for baseline absorbance (280 nm). Bound proteins were eluted by the addition of 4 column volumes of 0.4% TFA/80% acetonitrile. Elution fractions were combined, frozen, lyophilized, and stored at −20 °C. Neutravidin columns were prepared as described above. Serum proteins were digested in-solution to peptides with trypsin as follows: to 200 μg of protein 4 μg of sequencing grade trypsin (Promega) was added in ∼200 μl 50 mm ammonium bicarbonate; proteins were digested for 4 h at 37 °C. Total serum peptide digests were added directly to the neutravidin column. Nonbiotinylated peptides were removed with 10 column volumes of PBS-2% CHAPS, five column volumes of PBS and five column volumes of deionized water. Bound peptides were eluted by the addition of four column volumes of 0.4% TFA/80% acetonitrile. Elution fractions were combined, frozen, lyophilized, and stored at −20 °C. Mouse serum albumin and cytochrome c (1 mg/ml, 100 μl in pH 7.0 chelex-treated 100 mm HEPES buffer) were reacted with DODE (224 nmols, 10 μl ethanol) in the presence of vitamin C (10 mm) at 37 °C for 24 h. Proteins were filtered (regenerated cellulose 3000 Da MWCO; Amicon, Billerica, MA) to remove un-reacted DODE and vitamin C, and brought up in PBS to a concentration of 1 mg/ml. DODE modified protein samples (1 mg/ml) were incubated with ARP (10 mm) at a final volume of 1.0 ml in phosphate buffer, pH = 5–6. The reaction was stirred vigorously for 12 h at room temperature. The samples were filtered (regenerated cellulose 3000 Da MWCO; Amicon) to remove unreacted ARP. Neutravidin elutions of proteins from SJL mouse serum were digested in-solution with trypsin as follows: to 200 μg of protein 4 μg of sequencing grade trypsin (Promega) was added in ∼200 μl 25 mm ammonium bicarbonate; proteins were digested for 4 h at 37 °C. Digested proteins were desalted by SPE (Strata 50 μm, tri-Func, C18-E), dried and re-dissolved in 100 μl mobile phase A (described below) for SCX separation. Purified peptides were separated by SCX liquid chromatography using an Agilent 1100 LC system with a Polysulfoethyl A 100 × 4.6 mm, 5 μm column (Nest Group) and a flow rate of 0.25 ml/min. Mobile phase A was 10 mm sodium phosphate (Na2HPO4), 25% acetonitrile, pH = 2.8 and mobile phase B was 10 mm sodium phosphate (Na2HPO4), 25% acetonitrile, 0.4 m KCl, pH = 2.8. A linear gradient was as follows: 0% B from 0–10 min, 100% B at 70 min, 100% B at 75 min and 0% B at 80 min. Peptides were collected in 1-min fractions to give about 40–60 fractions per sample. These were each desalted by ZipTips® and redissolved in 5 μl 0.4% acetic acid for LC-electrospray ionization (ESI)-MS/MS analysis. The electrospray interface for this instrument uses a micro-tee (Upchurch Scientific, Oak Harbor, WA) with a 1-in. piece of platinum rod, inserted into one arm of the micro-tee, to supply the electrical connection. The electrospray voltage was typically 1600–1700 V applied just upstream of the column. Data-dependent MS/MS analysis was performed on the three most intense peaks in each full-scan spectrum, using multiply charged states (most of the nonpeptide background constituents are singly charged). Samples were spiked with 100 fmoles of vasoactive intestinal peptide fragment (amino acids 1–12) as the internal standard for calibration and mass accuracy. Accumulation time and pulsar frequency were maintained at 3 s and 6.99 s, respectively; the mass tolerance was 50 mmu. MS/MS was performed using nitrogen as the collision gas. Rolling collision energies were calculated as follows: z = 1, (collision energy) = 0.06*(m/z) + 8; z = 2, (collision energy) = 0.05*(m/z) + 5.6; z = 3, (collision energy) = 0.04*(m/z) + 6.7; z > 3, (collision energy) = 0.015*(m/z) + 25. Chromatography was performed using an Agilent 1100 capillary LC system with fused-silica capillary columns (75 μm i.d. × 360 μm o.d.; 14 cm length, tip 8 μm, New Objective, Woburn, MA) that were packed in-house with 5-μm C18 reverse-phase material (Vydac, Hesperia, CA). The flow rate from the pumps was 3–5 μl/min, and flow was split, before introduction of the sample onto the column, to 100–200 nL/min. Solvent A was 0.5% acetic acid in water and solvent B was 0.5% acetic acid in acetonitrile. A linear gradient was performed as follows: 2% B at 0 min, 2% B at 3 min, 65% B at 70 min, 80% B at 80 min, 80%B at 90 min, 2%B at 130 min, and 2% B at 170 min. Peak lists for the Applied Biosystems QSTAR Elite were generated as mgf files by Applied Biosystems Analyst QS 1.1 software. Database searches were carried out using Mascot version 2.2 (Matrix Science). For protein identification, either the NCBInr and/or the SwissProt mouse databases were searched. Parameters included: enzyme: trypsin; max missed cleavages = 2; fixed modifications carbamidomethyl (C) for two-dimensional gel analysis only; variable modifications of methionine oxidation (M); precursor tolerance was set at 0.1 Da; MS/MS fragment tolerance was set at 0.2 Da. Proteins were identified with three or more peptides. Significance of a protein match for Mascot was based on an expectation value of < 0.05 and a combined peptide score >50. For ARP-carbonyl modified peptides, the Mascot modification file was edited to add the DODE-ARP modification with elemental composition C24H37N5O7S (calculated monoisotopic mass 539.2414 Da) and specificity for lysine. In addition to the elemental composition of the modifying group, the Mascot modification file allows for the inclusion of neutral losses, used in scoring of a fragment ion spectrum and for the peptide neutral loss. The Mascot modification file also allows for ions to be ignored in scoring ("Ignore Masses"). Neutral losses and ignored ions were determined experimentally for ARP-DODE. Neutral-loss fragments included the entire modifying group, C24H37N5O7S (calculated monoisotopic mass 539.2414 Da) and the ARP portion of the modifying group, C12H21N5O4S (calculated monoisotopic mass 331.12124 Da). "Ignore Mass" ions included C10H15N2O2S (calculated monoisotopic mass 227.0854 Da), C10H19N4O2S (calculated monoisotopic mass 259.1229 Da), C12H19N4O3S (calculated monoisotopic mass 299.1178 Da) and C12H22N5O4S (calculated monoisotopic mass 332.1393 Da). All programming for the in silico filter program and the Biotin Identification Program was done in Python Programming Language (http://www.python.org). Python programs were written to be used on the Macintosh OS X operating system. Python text files for the in silico filter program and the Biotin Identification Program are available for view or download at: http://web.mit.edu/toxms/www/filters.htm. Proteins modified by carbonyls were identif

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