Biomedical subjects

Maria D Person

Publications and source records attributed to Maria D Person.

16 recordsLinked to original sources

PubMed2007

Protein expression profiles in the epidermis of cyclooxygenase-2 transgenic mice by 2-dimensional gel electrophoresis and mass spectrometry.

Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetone-treated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis.

Amino Acid Sequence↗

PubMed2006

Intracellular nucleotides act as critical prosurvival factors by binding to cytochrome C and inhibiting apoptosome.

Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apoptosis. This process can be reconstituted in vitro with the addition of CC and ATP or dATP to cell lysates. How physiological levels of nucleotides, normally at high mM concentrations, affect apoptosome activation remains unclear. Here we show that physiological levels of nucleotides inhibit the CC-initiated apoptosome formation and caspase-9 activation by directly binding to CC on several key lysine residues and thus preventing CC interaction with Apaf-1. We show that in various apoptotic systems caspase activation is preceded or accompanied by decreases in overall intracellular NTP pools. Microinjection of nucleotides inhibits whereas experimentally reducing NTP pools enhances both CC and apoptotic stimuli-induced cell death. Our results thus suggest that the intracellular nucleotides represent critical prosurvival factors by functioning as natural inhibitors of apoptosome formation and a barrier that cells must overcome the nucleotide barrier to undergo apoptosis cell death.

Apoptosis↗

PubMed2006

Inactivation of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor by 2-oxo-4-phenyl-3-butynoate.

Macrophage migration inhibitory factor (MIF) is an important immunoregulatory protein that has been implicated in several inflammatory diseases. MIF also has a phenylpyruvate tautomerase (PPT) activity, the role of which remains elusive in these biological activities. The acetylene compound, 2-oxo-4-phenyl-3-butynoate (2-OPB), has been synthesized and tested as a potential irreversible inhibitor of its enzymatic activity. Incubation of the compound with MIF results in the rapid and irreversible loss of the PPT activity. Mass spectral analysis established that the amino-terminal proline, previously implicated as a catalytic base in the PPT-catalyzed reaction, is the site of covalent modification. Inactivation of the PPT activity likely occurs by a Michael addition of Pro-1 to C-4 of the inhibitor. Attempts to crystallize the inactivated complex to confirm the structure of the adduct on the covalently modified Pro-1 by X-ray crystallography were not successful. Nor was it possible to unambiguously interpret electron density observed in the active sites of the native crystals soaked with the inhibitor. This may be due to crystal packing in that the side chain of Glu-16 from an adjacent trimer occupies one active site. However, this crystal contact may be partially responsible for the high-resolution quality of these MIF crystals. Nonetheless, because MIF is a member of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta structural motif and a catalytic Pro-1, 2-OPB may find general use as a probe of tautomerase superfamily members that function as PPTs.

Alkynes↗

PubMed2006

Protein fragment domains identified using 2D gel electrophoresis/MALDI-TOF.

We previously reported a protein expression profiling experiment conducted on human pancreatic tissues using 2D gel electrophoresis and mass spectrometry. Here, 18 spots that were identified in the gel at molecular weights more than 10 kDa lower than database values are characterized. The matrix-assisted laser desorption/ionization mass spectrometry coverage is sufficient to identify the protein region present in each spot. Most of the fragments correspond to processed chains and known structural or functional domains, which may result from limited proteolysis.

Amino Acid Sequence↗

PubMed2005

Inactivation of two diverse enzymes in the amidinotransferase superfamily by 2-chloroacetamidine: dimethylargininase and peptidylarginine deiminase.

The enzymes dimethylargininase [dimethylarginine dimethylaminohydrolase (DDAH); EC 3.5.3.18] and peptidylarginine deiminase (PAD; EC 3.5.3.15) catalyze hydrolysis of substituted arginines. Due to their role in normal physiology and pathophysiology, both enzymes have been identified as potential drug targets, but few useful inhibitors have been reported. Here, we find that 2-chloroacetamidine irreversibly inhibits both DDAH from Pseudomonas aeruginosa and human PAD4 in a time- and concentration-dependent manner, despite the nonoverlapping substrate specificities and low levels of amino acid identity of their catalytic domains. Substrate protection experiments indicate that inactivation occurs by modification at the active site, albeit with modest affinity. Mass spectral analysis demonstrates that irreversible inactivation of DDAH occurs through selective formation of a covalent thioether bond with the active-site Cys249 residue. The mechanism of inactivation by 2-chloroacetamidine is analogous to that of chloromethyl ketones, a set of inhibitors that have found wide application because of their specific covalent modification of active-site residues in serine and cysteine proteases. Likewise, 2-chloroacetamidine may potentially find wide applicability as a general pharmacophore useful in delineating characteristics of the amidinotransferase superfamily.

Amidines↗

PubMed2005

Characterization of a transient covalent adduct formed during dimethylarginine dimethylaminohydrolase catalysis.

Dimethylarginine dimethylaminohydrolase (DDAH) regulates the concentrations of human endogenous inhibitors of nitric oxide synthase, N(omega)-methyl-l-arginine (NMMA), and asymmetric N(omega),N(omega)-dimethyl-l-arginine (ADMA). Pharmacological regulation of nitric oxide synthesis is an important goal, but the catalytic mechanism of DDAH remains largely unexplored. A DDAH from Pseudomonas aeruginosa was cloned, and asymmetrically methylated arginine analogues were shown to be the preferred substrates, with ADMA displaying a slightly higher k(cat)/K(M) value than NMMA. DDAH is similar to members of a larger superfamily of guanidino-modifying enzymes, some of which have been shown to use an S-alkylthiouronium intermediate during catalysis. No covalent intermediates were found to accumulate during steady-state turnover reactions of DDAH with NMMA or ADMA. However, identification of a new substrate with an activated leaving group, S-methyl-l-thiocitrulline (SMTC), enabled acid trapping and ESI-MS characterization of a transient covalent adduct with a mass of 158 +/- 10 Da that accumulates during steady-state turnover. Subsequent trapping, proteolysis, peptide mapping and fragmentation by mass spectrometry, and site-directed mutagenesis demonstrated that this covalent adduct was attached to an active site residue and implicates Cys249 as the catalytic nucleophile required for intermediate formation. The use of covalent catalysis clearly links DDAH to this superfamily of enzymes and suggests that an S-alkylthiouronium intermediate may be a conserved feature in their mechanisms.

Acids↗

PubMed2005

Ribosomal protein S2 is a substrate for mammalian PRMT3 (protein arginine methyltransferase 3).

PRMT3 (protein arginine methyltransferase 3) is one of four type I arginine methyltransferases that catalyse the formation of asymmetric dimethylarginine. PRMT3 is unique in that its N-terminus harbours a C2H2 zinc-finger domain that is proposed to confer substrate specificity. In addition, PRMT3 is the only type I enzyme that is restricted to the cytoplasm. Known in vitro substrates for PRMT3 include GST-GAR (a glutathione S-transferase fusion protein containing the glycine- and arginine-rich N-terminal region of fibrillarin), Sam68 (Src-associated substrate during mitosis 68 kDa) and PABP-N1 [poly(A)-binding protein-N1; PABP2]. Here we report the identification of an in vivo substrate for mammalian PRMT3. We found that FLAG-tagged PRMT3 can 'pull down' a protein with a molecular mass of 30 kDa from HeLa cell extracts. MS identified this PRMT3-interacting protein as rpS2 (ribosomal protein S2). In vitro studies showed that the zinc-finger domain of PRMT3 is necessary and sufficient for binding to rpS2. In addition, rpS2 is methylated by PRMT3 in vitro and is also methylated in cell lines. Deletion analysis of the rpS2 amino acid sequence identified a N-terminal Arg-Gly repeat as the methylation site. Furthermore, both PRMT3 and rpS2 co-sediment with free ribosomal subunits. These studies implicate PRMT3 in ribosomal function and in the regulation of protein synthesis.

Animals↗

PubMed2005

Alkylation of cytochrome c by (glutathion-S-yl)-1,4-benzoquinone and iodoacetamide demonstrates compound-dependent site specificity.

The reaction of cytochrome c with the electrophilic compounds (glutathion-S-yl)-1,4-benzoquinone (GSBQ) and iodoacetamide was studied using mass spectrometry. GSBQ is a nephrotoxic quinol-thioether metabolite of benzoquinone, while iodoacetamide is an alkylating agent targeting cysteine thiols. Both chemicals formed covalent adducts with cytochrome c. GSBQ formed adducts with cytochrome c at pH 6 on several histidine and lysine residues. At a pH >7, the initial product rearranged to a disubstituted cyclic quinone species preferentially found at two sites on the protein, Lys25-Lys27 and Lys86-Lys87, via quinol amine linkages. These two sites were previously determined to be the targets of benzoquinone adduct formation [Person et al. (2003) Chem. Res. Toxicol. 16, 598-608]. Cyclic reaction products are preferentially formed at two sites on the protein because of the presence of multiple basic residues in a conformationally flexible region whereas noncyclic products bind to a broad spectrum of available lysine and histidine nucleophiles. Iodoacetamide was a less selective alkylating agent able to form adducts on the majority of the nucleophilic sites of the protein. MS/MS spectra were used to identify signature ions for GSBQ-adducted peptides from the characteristic fragmentation patterns. Neutral losses of the 129 Da gamma-glutamate residue and of the 273 Da glutathione moiety were found in both cysteine thiol- and lysine amine-linked GSBQ adduct MS/MS. Characteristic fragment ions were used in conjunction with the scoring algorithm for spectral analysis to search for adducted species present at low levels in the sample, and the analysis is applicable generally to detection of glutathione conjugates by MS/MS. Parallel analysis using matrix-assisted laser desorption/ionization-MS to compare spectra of control and treated samples allowed identification of peptide adducts formed by direct addition of GSBQ and by the subsequent loss of the glutathione moiety in a pH-dependent cyclization reaction.

Algorithms↗

PubMed2004

Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.

Pancreatic cancer is a rapidly fatal disease, and there is an urgent need for early detection markers and novel therapeutic targets. The current study has used a proteomic approach of two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) to identify differentially expressed proteins in six cases of pancreatic adenocarcinoma, two normal adjacent tissues, seven cases of pancreatitis, and six normal pancreatic tissues. Protein extracts of individual sample and pooled samples of each type of tissues were separated on 2D gels using two different pH ranges. Differentially expressed protein spots were in-gel digested and identified by MS. Forty proteins were identified, of which five [i.e., alpha-amylase; copper zinc superoxide dismutase; protein disulfide isomerase, pancreatic; tropomyosin 2 (TM2); and galectin-1] had been associated previously with pancreatic disease in gene expression studies. The identified proteins include antioxidant enzymes, chaperones and/or chaperone-like proteins, calcium-binding proteins, proteases, signal transduction proteins, and extracellular matrix proteins. Among these proteins, annexin A4, cyclophilin A, cathepsin D, galectin-1, 14-3-3zeta, alpha-enolase, peroxiredoxin I, TM2, and S100A8 were specifically overexpressed in tumors compared with normal and pancreatitis tissues. Differential expression of some of the identified proteins was further confirmed by Western blot analyses and/or immunohistochemical analysis. These results show the value of a proteomic approach in identifying potential markers for early diagnosis and therapeutic manipulation. The newly identified proteins in pancreatic tumors may eventually serve as diagnostic markers or therapeutic targets.

Biomarkers, Tumor↗

PubMed2004

Grp78 is essential for 11-deoxy-16,16-dimethyl PGE2-mediated cytoprotection in renal epithelial cells.

11-Deoxy-16,16-dimethyl PGE(2) (DDM-PGE(2)) protects renal proximal tubule epithelial cells (LLC-PK(1)) against the toxicity induced by 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic and nephrocarcinogenic metabolite of hydroquinone. We have now determined the ability of DDM-PGE(2) to protect against other renal toxicants and report that DDM-PGE(2) only protects against oncotic cell death, induced by H(2)O(2), iodoacetamide, and TGHQ, but not against apoptotic cell death induced by cisplatin, mercuric chloride, or tumor necrosis factor-alpha. DDM-PGE(2)-mediated cytoprotection is associated with the upregulation of at least five proteins, including the major endoplasmic reticulum (ER) chaperone glucose-regulated protein 78 (Grp78). To elucidate the role of Grp78 in oncotic cell death, we used LLC-PK(1) cells in which induction of grp78 expression was disrupted by stable expression of an antisense grp78 RNA (pkASgrp78). As anticipated, DDM-PGE(2) failed to induce Grp78 in pkASgrp78 cells, with a concomitant inability to provide cytoprotection. In contrast, DDM-PGE(2) induced Grp78 and afforded cytoprotection against H(2)O(2), iodoacetamide, and TGHQ in empty vector transfected cells (pkNEO). These data suggest that Grp78 plays an essential role in DDM-PGE(2)-mediated cytoprotection. Moreover, TGHQ-induced p38 MAPK activation is disrupted under conditions of a compromised ER stress response in pkASgrp78 cells, which likely contributes to the loss of cytoprotection. Finally, using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we found that DDM-PGE(2) induced several proteins in pkNEO cells, but not in pkASgrp78 cells, including retinol-binding protein, myosin light chain, and heat shock protein 27. The findings suggest that additional proteins may act in concert with Grp78 during DDM-PGE(2)-mediated cytoprotection against oncotic cell death.

Animals↗

PubMed2004

Cloning, expression, and characterization of a cis-3-chloroacrylic acid dehalogenase: insights into the mechanistic, structural, and evolutionary relationship between isomer-specific 3-chloroacrylic acid dehalogenases.

The gene encoding the cis-3-chloroacrylic acid dehalogenase (cis-CaaD) from coryneform bacterium strain FG41 has been cloned and overexpressed, and the enzyme has been purified to homogeneity and subjected to kinetic and mechanistic characterization. Kinetic studies show that cis-CaaD processes cis-3-haloacrylates, but not trans-3-haloacrylates, with a turnover number of approximately 10 s(-1). The product of the reaction is malonate semialdehyde, which was confirmed by its characteristic 1H NMR spectrum. The enzyme shares low but significant sequence similarity with the previously studied trans-3-chloroacrylic acid dehalogenase (CaaD) and with other members of the 4-oxalocrotonate tautomerase (4-OT) family. While 4-OT and CaaD function as homo- and heterohexamers, respectively, cis-CaaD appears to be a homotrimeric protein as assessed by gel filtration chromatography. On the basis of the known three-dimensional structures and reaction mechanisms of CaaD and 4-OT, a sequence alignment implicated Pro-1, Arg-70, Arg-73, and Glu-114 as important active-site residues in cis-CaaD. Subsequent site-directed mutagenesis experiments confirmed these predictions. The acetylene compounds, 2-oxo-3-pentynoate and 3-bromo- and 3-chloropropiolate, were processed by cis-CaaD to products consistent with an enzyme-catalyzed hydration reaction previously established for CaaD. Hydration of 2-oxo-3-pentynoate afforded acetopyruvate, while the 3-halopropiolates became irreversible inhibitors that modified Pro-1. The results of this work revealed that cis-CaaD and CaaD have different primary and quaternary structures, and display different substrate specificity and catalytic efficiencies, but likely share a highly conserved catalytic mechanism. The mechanism may have evolved independently because sequence analysis indicates that cis-CaaD is not a 4-OT family member, but represents the first characterized member of a new family in the tautomerase superfamily that probably resulted from an independent duplication of a 4-OT-like sequence. The discovery of a fifth family of enzymes within this superfamily further demonstrates the diversity of activities and structures that can be created from 4-OT-like sequences.

Amino Acid Sequence↗

PubMed2003

Reactions of trans-3-chloroacrylic acid dehalogenase with acetylene substrates: consequences of and evidence for a hydration reaction.

Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is trans-3-chloroacrylic acid dehalogenase (CaaD), which converts the trans-isomers of 3-bromo- and 3-chloroacrylate to malonate semialdehyde. Sequence analysis suggested a relationship between the heterohexameric CaaD and the bacterial isomerase, 4-oxalocrotonate tautomerase (4-OT), thereby distinguishing CaaD from a number of dehalogenases whose mechanisms proceed through an alkyl- or aryl-enzyme intermediate. In this study, the genes for the alpha- and beta-subunits of CaaD have been synthesized using a polymerase chain reaction-based strategy, cloned into separate plasmids, and the proteins expressed and purified as the functional heterohexamer. Subsequently, the product of the reaction was confirmed to be malonate semialdehyde by (1)H and (13)C NMR spectroscopy, and kinetic constants were determined using a UV spectrophotometric assay. In view of the proposed hydrolytic nature of the CaaD-catalyzed reaction, three acetylene compounds were investigated as substrates for the enzyme. One compound, 2-oxo-3-pentynoate, a potent active site-directed irreversible inhibitor of 4-OT, is a substrate for CaaD, and was processed to acetopyruvate with kinetic constants similar to those determined for the trans-isomers of 3-bromo- and 3-chloroacrylate. The remaining two compounds, 3-bromo- and 3-chloropropiolic acid, were transformed into potent irreversible inhibitors of CaaD. The inactivation observed for 3-bromopropiolic acid is due to the covalent modification of Pro-1 of the beta-subunit. The results provide evidence for a hydratase activity and set the stage to use the 3-halopropiolic acids as ligands in inactivated CaaD complexes that can be studied by X-ray crystallography.

Acetylene↗

PubMed2003

Annexin II expression is reduced or lost in prostate cancer cells and its re-expression inhibits prostate cancer cell migration.

While studying Bim, a BH3-only proapoptotic protein, we identified an approximately 36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven prostate cancer cell lines. The approximately 36 kDa protein was subsequently identified as annexin II by proteomic approach and confirmed by Western blotting using an annexin II-specific antibody. Conventional and 2D SDS-PAGE, together with Western blotting, also revealed reduced or lost expression of annexin I in prostate cancer cells. Subcellular localization studies revealed that in NHP cells, annexin II was distributed both in the cytosol and underneath the plasma membrane, but not on the cell surface. Prostate cancer cells showed reduced levels as well as altered expression patterns of annexin II. Since annexins play important roles in maintaining Ca(2+) homeostasis and regulating the cytoskeleton and cell motility, we hypothesized that the reduced or lost expression of annexin I/II might promote certain aggressive phenotypes of prostate cancer cells. In subsequent experiments, we indeed observed that restoration of annexin II expression inhibited the migration of the transfected prostate cancer cells without affecting cell proliferation or apoptosis. Hence, our results suggest that annexin II, and, likely, annexin I, may be endogenous suppressors of prostate cancer cell migration and their reduced or lost expression may contribute to prostate cancer development and progression.

Amino Acid Sequence↗

PubMed2003

11-Deoxy,16,16-dimethyl prostaglandin E2 induces specific proteins in association with its ability to protect against oxidative stress.

Prostaglandins (PGs) act locally to maintain cellular homeostasis and stimulate stress response signaling pathways. These cellular effects are diverse and are tissue-dependent. PGE(2), and the synthetic analogue, 11-deoxy,16,16-dimethyl PGE(2) (DDM-PGE(2)), protect renal proximal tubular epithelial (LLC-PK1) cells against cellular injury induced by the potent nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, 2,3,5-tris-(glutathion-S-yl)hydroquinone. Although this cytoprotective response (in LLC-PK1 cells) is mediated through a thromboxane or thromboxane-like receptor coupled to AP-1 signaling pathways, the mechanism of cytoprotection is unknown. In this study, we utilized HPLC-electrospray ionization tandem mass spectrometric (ESI MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometric (MALDI TOF) analysis of proteins isolated from DDM-PGE(2)-stimulated LLC-PK1 cells to identify candidate cytoprotective proteins. DDM-PGE(2) selectively stimulated the synthesis of several proteins in LLC-PK1 cells. Peptide sequencing by ESI-MS/MS of in-gel tryptic protein digests revealed the identity of eight proteins: endothelial actin binding protein, myosin, elongation factor 2 (EF-2), elongation factor 1alpha-1 (EF-1alpha), heat shock protein 90beta (HSP90beta), glucose-regulated protein 78 (GRP 78), membrane-organizing extension spike protein, and actin. Both ESI-MS/MS and MALDI-MS analysis resulted in the same protein identification. Western analysis confirmed the temporal induction of the majority of these proteins, including EF-2, EF-1alpha, HSP90beta, GRP78, and actin. The collective expression of these proteins suggests that DDM-PGE(2)-mediated cytoprotection may involve alterations in cytoskeletal organization and/or stimulation of an endoplasmic reticulum (ER) stress response. The present studies provide insights into potential downstream targets of PG signaling.

16,16-Dimethylprostaglandin E2↗

PubMed2003

Comparative identification of prostanoid inducible proteins by LC-ESI-MS/MS and MALDI-TOF mass spectrometry.

Protein identification by MS is well-established. Mixtures of proteins from cell extracts are separated by either one- or two-dimensional gel electrophoresis, and specific bands or spots are subjected to in-gel digestion and subsequent analysis by MS. The two most common types of ionization used in MS are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). When ESI is used, the sample is typically analyzed by inline HPLC-ESI-MS/MS with fragmentation of individual digest peptides, followed by database comparison between theoretical and experimental fragmentation patterns. MALDI-MS analysis is based on peptide mass mapping, with mass measurements of the digest peptides searched against a database of theoretical digests. We give here the results of a comparison between ESI-ion trap and MALDI-TOF (time-of-flight) analysis of 11-deoxy,16,16-dimethyl prostaglandin E(2) (DDM-PGE(2)) inducible proteins. Individual peptides identified by the two techniques differed, in general, but the resulting protein identification was the same. Slightly higher coverage of each protein was obtained by MALDI-TOF, but the MS/MS data were more definitive by requiring fewer peptides to assign a positive identification. Both methods effectively identified two proteins in the same gel band. The samples here are derived from a renal epithelial cell line (LLC-PK(1)) established from the New Hampshire minipig, a species poorly represented in the current database, and strategies and limitations for analyzing such species are discussed.

Amino Acid Sequence↗

PubMed2003

An integrated approach to identifying chemically induced posttranslational modifications using comparative MALDI-MS and targeted HPLC-ESI-MS/MS.

Identification of multiple and novel posttranslational modifications remains a major challenge in proteomics. The present approach uses comparative analysis by matrix-assisted laser/desorption ionization (MALDI) MS of proteolytic digests from control and treated proteins to target differences due to modifications, without initial assumption as to type or residue localization. Differences between modified and unmodified digest MS spectra highlight peptides of interest for subsequent tandem mass spectrometry (MS/MS) analysis. Targeted HPLC-electrospray ionization (ESI)-MS/MS is then used to fragment peptides, and manual de novo sequencing is used to determine the amino acid sequence and type of modification. This strategy for identifying posttranslational modifications in an unbiased manner is particularly useful for finding modifications produced by exogenous chemicals. Successful characterization of chemically induced posttranslational modifications and novel chemical adducts is given as an example of the use of this strategy. Histone H4 from butyrate-treated LLC-PK1 cells is separated on a gel into bands representing different overall charge state. Bands are analyzed by comparative MALDI-MS and LC-MS/MS to identify the sites of methylation and acetylation. Previous attempts to identify chemically adducted proteins in vivo have been unsuccessful in part due to a lack of understanding of the final adduct form. Cytochrome c is adducted in vitro with benzoquinone, an electrophilic metabolite of benzene capable of interacting with nucleophilic sites within proteins. De novo sequencing identifies a novel cyclized diquinone adduct species as the major reaction product, targeting Lys and His residues at two specific locations on the protein surface. This unpredicted reaction product is characterized using our unbiased methods for detection and demonstrates the important influence of protein structure on chemical adduction.

Acetylation↗