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1.  Proteomic Analysis of Laser Microdissected Melanoma Cells from Skin Organ Cultures 
Journal of proteome research  2010;9(7):3656-3663.
Gaining insights into the molecular events that govern the progression from melanoma in situ to advanced melanoma, and understanding how the local microenvironment at the melanoma site influences this progression, are two clinically pivotal aspects that to date are largely unexplored. In an effort to identify key regulators of the crosstalk between melanoma cells and the melanoma-skin microenvironment, primary and metastatic human melanoma cells were seeded into skin organ cultures (SOCs), and grown for two weeks. Melanoma cells were recovered from SOCs by laser microdissection and whole-cell tryptic digests analyzed by nanoflow liquid chromatography-tandem mass spectrometry with an LTQ-Orbitrap. The differential protein abundances were calculated by spectral counting, the results of which provides evidence that cell-matrix and cell-adhesion molecules that are upregulated in the presence of these melanoma cells recapitulate proteomic data obtained from comparative analysis of human biopsies of invasive melanoma and a tissue sample of adjacent, non-involved skin. This concordance demonstrates the value of SOCs for conducting proteomic investigations of the melanoma microenvironment.
doi:10.1021/pr100164x
PMCID: PMC3733114  PMID: 20459140
melanoma; proteomics; skin organ culture
2.  CLT1 Targets Angiogenic Endothelium through CLIC1 and Fibronectin 
Angiogenesis  2011;15(1):115-129.
Angiogenesis is important for tumor growth and metastasis. CLT1 (CGLIIQKNEC), a peptide that binds to tumor interstitial spaces in the presence of fibrin-fibronectin, has structural similarity to the anti-angiogenic β-sheet peptides anastellin and anginex. This similarity is reflected in the ability of CLT1 to form co-aggregates with fibronectin that induce an unfolded protein response and cause autophagic cell death in proliferating endothelial cells. CLT1 cytotoxicity is mediated at least in parts by a novel CLT1 binding protein, Chloride Intracellular Channel 1 (CLIC1), which promotes internalization of CLT1-fibronectin co-aggregates in a mechanism that depends on the LIIQK amino acid sequence of CLT1. LIIQK encompasses amino acid residues relevant for CLT1 binding to CLIC1 and in addition, facilitates the formation of CLT1-fibronectin co-aggregates, which in turn promote translocation of CLIC1 to the endothelial cell surface through ligation of integrin αvβ3. Paralleling the in vitro results, we found that CLT1 co-localizes with CLIC1 and fibronectin in angiogenic blood vessels in vivo, and that CLT1 treatment inhibited angiogenesis and tumor growth. Our findings show that CLT1 is a new anti-angiogenic compound, and its mechanism of action is to form co-aggregates with fibronectin, which bind to angiogenic endothelial cells through integrins, become internalized through CLIC1 and elicit a cytotoxic unfolded protein response. The simple structure and high potency of CLT1 make it a potentially useful compound for anti-angiogenic treatments.
doi:10.1007/s10456-011-9247-8
PMCID: PMC3320048  PMID: 22203240
CLT1; fibronectin; chloride intracellular channel 1; angiogenesis; integrin
3.  Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes 
Journal of proteome research  2012;11(10):4983-4991.
ATM is a protein kinase that initiates a well-characterized signaling cascade in cells exposed to ionizing radiation (IR). However, the role for ATM in coordinating critical protein interactions and subsequent exchanges within DNA damage response (DDR) complexes is unknown. We combined SILAC-based tandem mass spectrometry and a subcellular fractionation protocol to interrogate the proteome of irradiated cells treated with or without the ATM kinase inhibitor KU55933. We developed an integrative network analysis to identify and prioritize proteins that were responsive to KU55933, specifically in chromatin, and that were also enriched for physical interactions with known DNA repair proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as strong candidates. Using fluorescence recovery after photobleaching, we found that the exchange of GFP-53BP1 in DDR complexes decreased with KU55933. Further, we found that ANXA1 knockdown sensitized cells to IR via a mechanism that was not potentiated by KU55933. Our study reveals a role for ATM kinase activity in the dynamic exchange of proteins in DDR complexes and identifies a role for ANXA1 in cellular radioprotection.
doi:10.1021/pr3005524
PMCID: PMC3495236  PMID: 22909323
DNA damage response; ATM; 53BP1
4.  Identification of Potential Protein Targets of Isothiocyanates by Proteomics 
Chemical research in toxicology  2011;24(10):1735-1743.
Isothiocyanates (ITCs), such as phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), are effective cancer chemopreventive compounds. It is believed that a major mechanism for the cancer preventive activity of ITCs is through induction of cell cycle arrest and apoptosis. However, the upstream molecular targets of ITCs have been underexplored until recently. To identify proteins that are covalently modified by ITCs, human non-small cell lung cancer A549 cells were treated with 14C-PEITC and 14C-SFN and the cell lysates were extracted for analysis by 2-D gel electrophoresis and mass spectrometry. After superimposing the colloidal Coomassie blue protein staining pattern with the pattern of radioactivity obtained from X-ray films, it was clear that only a small fraction of cellular proteins contained radioactivity, presumably resulting from selective binding with PEITC or SFN via thiocarbamation. More than 30 proteins with a variety of biological functions were identified with high confidence. Here we report the identities of these potential ITC target proteins and discuss their biological relevance. The discovery of the protein targets may facilitate studies of the mechanisms by which ITCs exert their cancer preventive activity and provide molecular basis for designing more efficacious ITC compounds.
doi:10.1021/tx2002806
PMCID: PMC3493163  PMID: 21838287
5.  The RNA-binding motif 45 (RBM45) protein accumulates in inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) patients 
Acta Neuropathologica  2012;124(5):717-732.
RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer’s disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-012-1045-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-012-1045-x
PMCID: PMC3472056  PMID: 22993125
Amyotrophic lateral sclerosis; Frontotemporal lobar degeneration; TDP-43; RNA-binding protein; RBM45; C9ORF72
6.  Lung Cancer Serum Biomarker Discovery Using Label Free LC-MS/MS 
Introduction
Lung cancer remains the leading cause of cancer-related death with poor survival due to the late stage at which lung cancer is typically diagnosed. Given the clinical burden from lung cancer, and the relatively favorable survival associated with early stage lung cancer, biomarkers for early detection of lung cancer are of important potential clinical benefit.
Methods
We performed a global lung cancer serum biomarker discovery study using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a set of pooled non-small cell lung cancer (NSCLC) case sera and matched controls. Immunoaffinity subtraction was used to deplete the top most abundant serum proteins; the remaining serum proteins were subjected to trypsin digestion and analyzed in triplicate by LC-MS/MS. The tandem mass spectrum data were searched against the human proteome database and the resultant spectral counting data were used to estimate the relative abundance of proteins across the case/control serum pools. The spectral counting derived abundances of some candidate biomarker proteins were confirmed with multiple reaction monitoring MS assays.
Results
A list of 49 differentially abundant candidate proteins was compiled by applying a negative binomial regression model to the spectral counting data (p<0.01). Functional analysis with Ingenuity Pathway Analysis tools showed significant enrichment of inflammatory response proteins, key molecules in cell-cell signaling and interaction network and differential physiological responses for the two common NSCLC subtypes.
Conclusions
We identified a set of candidate serum biomarkers with statistically significant differential abundance across the lung cancer case/control pools which, when validated, could improve lung cancer early detection.
doi:10.1097/JTO.0b013e31820c312e
PMCID: PMC3104087  PMID: 21304412
Lung cancer; serum biomarkers; LC-MS/MS
7.  A UPLC-MS/MS assay of the “Pittsburgh Cocktail”: six CYP probe-drug/metabolites from human plasma and urine using stable isotope dilution 
The Analyst  2010;136(3):605-612.
The efficiency of drug metabolism by a single enzyme can be measured as the fractional metabolic clearance which can be used as a measure of whole body activity for that enzyme. Measurement of activity of multiple enzymes simultaneously is feasible using a cocktail approach however analytical approach using different assays for drug probes can be cumbersome. A quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)-based method for the rapid measurement of six cytochrome P450 (CYP) probe drugs and their relevant metabolites, is described. The six specific probe substrates/metabolites, are caffeine/paraxanthine (CYP1A2), flurbiprofen/4′-hydroxyflurbiprofen (CYP2C9), mephenytoin/4′-hydroxymephenytoin (CYP2C19), debrisoquine/4-hydroxydebrisoquine (CYP2D6), chlorzoxazone/6′-hydroxychlorzoxazone (CYP2E1) and dapsone/N-monoacetyldapsone (NAT2). These probes were quantified by stable isotope dilution from plasma and urine. The present workflow provides a robust, fast and sensitive assay for the “Pittsburgh Cocktail”, and has been successfully applied to a clinical phenotyping study of liver disease. A representative group of 17 controls and patients with chronic liver disease were administered orally caffeine (100mg), chlorzoxazone (250mg), debrisoquine (10mg), mephenytoin (100mg) flurbiprofen (50mg) and dapsone (100mg). Urine (0 through 8 h) and plasma (4 and 8 h) samples were analyzed for drug/metabolite amounts by stable isotope dilution UPLC-MS/MS. The phenotypic activity of drug metabolizing enzymes was investigated with 17 patient samples. Selected reaction monitoring (SRM) was optimized for each drug and metabolite. In the method developed, analytes were resolved by reversed-phase by development of a gradient using a water/methanol solvent system. SRM of each analyte was performed in duplicate on a triple quadrupole mass spectrometer utilizing an 8 min analytical method each, one with the source operating in the positive mode and one in the negative mode, using the same solvent system. This method enabled quantification of each drug (caffeine, chlorzoxazone, debrisoquine, mephenytoin, flurbiprofen, and dapsone) and its resulting primary metabolite in urine or plasma in patient samples. The method developed and the data herein demonstrate a robust quantitative assay to examine changes in CYP enzymes both independently or as part of a cocktail. The clinical use of a combination of probe drugs with UPLC-MS/MS is a highly efficient tool for the assessment of CYP enzyme activity in liver disease.
doi:10.1039/c0an00643b
PMCID: PMC3115584  PMID: 21107456
CYP; Mass spectrometry; stable isotope dilution; phenotyping; drug cocktail
8.  Identification of the Homeobox Protein Prx1 (MHox, Prrx-1) as a Regulator of Osterix Expression and Mediator of Tumor Necrosis Factor α Action in Osteoblast Differentiation 
Tumor necrosis factor α (TNF-α) promotes bone loss and inhibits bone formation. Osterix (Osx, SP7) is a transcription factor required for osteoblast (OB) differentiation because deletion results in a cartilaginous skeleton. We previously described a TNF suppressor element in the Osx promoter that was used to isolate nuclear proteins mediating TNF inhibition of OB differentiation. Nuclear extracts from TNF-treated pre-OBs were incubated with the TNF suppressor element for protein pull-down, and tryptic fragments were analyzed by mass spectrometry. Chromatin immunoprecipitation (ChIP) assay confirmed eight bound transcription factors. One protein, the paired related homeobox protein (Prx1), had been shown previously to have a critical role in limb bud formation and skeletal patterning. PCR revealed Prx1 expression in primary stromal cells (MSCs), C3H10T1/2 cells, and MC3T3 preosteoblasts. TNF stimulated a 14-fold increase in mRNA for Prx1, rapid cell accumulation in MC3T3 cells, and expression in periosteal and trabecular lining cells in vivo. Transient expression of Prx inhibited transcription of Osx and RUNX2. Expression of the Prx1b isoform or Prx2 decreased Osx and RUNX2 mRNA and OB differentiation in preosteoblasts. Silencing of Prx1 with siRNA abrogated TNF suppression of Osx mRNA and increased basal Osx expression. Electrophoretic mobility shift revealed Prx1b as the preferred isoform binding the Osx promoter. These results identify the homeobox protein Prx1 as an obligate mediator of TNF inhibition of Osx and differentiation of OB progenitors. Activation of Prx1 by TNF may contribute to reduced bone formation in inflammatory arthritis, menopause, and aging. © 2011 American Society for Bone and Mineral Research.
doi:10.1002/jbmr.203
PMCID: PMC3179318  PMID: 20683885
OSTEOBLASTS; BONE; TNF; PRX1; MHOX
9.  Analysis of PSPHL as a Candidate Gene Influencing the Racial Disparity in Endometrial Cancer 
Endometrial cancer is the most commonly diagnosed gynecologic malignancy in the United States. A well recognized disparity by race in both incidence and survival outcome exists for this cancer. Specifically Caucasians are about two times more likely to develop endometrial cancer than are African-Americans. However, African-American women are more likely to die from this disease than are Caucasians. The basis for this disparity remains unknown. Previous studies have identified differences in the types and frequencies of gene mutations among endometrial cancers from Caucasians and African-Americans suggesting that the tumors from these two groups might have differing underlying genetic defects. We performed a gene expression microarray study in an effort to identify differentially expressed transcripts between African-American and Caucasian women’s endometrial cancers. Our gene expression screen identified a list of potential biomarkers that are differentially expressed between these two groups of cancers. Of these we identified a poorly characterized transcript with a region of homology to phospho serine phosphatase (PSPH) and designated phospho serine phosphatase like (PSPHL) as the most differentially over-expressed gene in cancers from African-Americans. We further clarified the nature of expressed transcripts. Northern blot analysis confirmed the message was limited to a transcript of under 1 kB. Sequence analysis of transcripts confirmed two alternate open reading frame (ORF) isoforms due to alternative splicing events. Splice specific primer sets confirmed both isoforms were differentially expressed in tissues from Caucasians and African-Americans. We further examined the expression in other tissues from women to include normal endometrium, normal and malignant ovary. In all cases PSPHL expression was more often present in tissues from African-Americans than Caucasians. Our data confirm the African-American based expression of the PSPHL transcript in endometrial cancer and also identify its expression in other tissues from African-Americans including ovary and ovarian cancer. PSPHL represents a candidate gene that might influence the observed racial disparity in endometrial and other cancers.
doi:10.3389/fonc.2012.00065
PMCID: PMC3389395  PMID: 22783543
endometrial cancer; PSPHL; racial disparity
10.  Lung Cancer Serum Biomarker Discovery Using Glycoprotein Capture and Liquid Chromatography Mass Spectrometry 
Journal of proteome research  2010;9(12):6440-6449.
Targeted glycoproteomics represents an attractive approach for conducting peripheral blood based cancer biomarker discovery due to the well-known altered pattern of protein glycosylation in cancer and the reduced complexity of the resultant glycoproteome. Here we report its application to a set of pooled non-small cell lung cancer (NSCLC) case sera (9 adenocarcinoma and 6 squamous cell carcinoma pools from 54 patients) and matched controls pools, including 8 clinical control pools with computed tomography detected nodules but being non-malignant as determined by biopsy from 54 patients, and 8 matched healthy control pools from 106 cancer-free subjects. The goal of the study is to discover biomarkers which may enable improved early detection and diagnosis of lung cancer. Immunoaffinity subtraction was used to first deplete the top most abundant serum proteins; the remaining serum proteins were then subjected to hydrazide chemistry based glycoprotein capture and enrichment. Hydrazide resin in situ trypsin digestion was used to release non-glycosylated peptides. Formerly N-linked glycosylated peptides were released by peptide-N-glycosidase F (PNGase F) treatment and were subsequently analyzed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS). A MATLAB® based in-house tool was developed to facilitate retention time alignment across different LC-MS/MS runs, determination of precursor ion m/z values and elution profiles, and the integration of mass chromatograms based on determined parameters for identified peptides. A total of 38 glycopeptides from 22 different proteins were significantly differentially abundant across the case/control pools (P<0.01, Student’s t test) and their abundances led to a near complete separation of case and control pools based on hierarchical clustering. The differential abundances of three of these candidate proteins were verified by commercially available ELISAs applied in the pools. Strong positive correlations between glycopeptide mass chromatograms and ELISA-measured protein abundance was observed for all of the selected glycoproteins.
doi:10.1021/pr100696n
PMCID: PMC3184639  PMID: 20931982
Lung cancer; serum biomarkers; glycoproteomics; LC-MS/MS; mass chromatogram
11.  Novel Surgical Approaches for Sampling the Ovarian Surface Epithelium and Proximal Fluid Proteome 
Journal of proteome research  2010;9(11):6071-6076.
The pathogenesis of ovarian, fallopian tube, and peritoneal cancers has been difficult to elucidate despite intense effort. Recently, though, the care of women felt to be at high risk due to a strong family history of breast and/or ovarian cancer or a known germline BRCA1 or BRCA2 mutation has provided potential insight into the development of these malignancies. Risk-reducing surgical removal of the fallopian tubes and ovaries, called risk-reducing bilateral salpingo-oopherectomy (RRBSO), is commonly performed as a laparoscopic procedure to minimize recovery time. We describe here an optimized surgical sampling workflow for analyzing the proteomes of peritoneal, fallopian tube, and ovarian surface epithelial (OSE) specimens collected at the time of laparoscopic RRBSO, a technique which has not been described previously. This methodology presents a unique opportunity for closer examination of the proteomic alterations in the tissues at risk for malignant transformation in women with an inherited susceptibility to ovarian, fallopian tube, and peritoneal cancer development.
doi:10.1021/pr100451f
PMCID: PMC3190590  PMID: 20873867
ovarian cancer; ovarian surface epithelium; fallopian tube; proteomics; mass spectrometry
12.  Proteomic Analysis of Ovarian Cancer Proximal Fluids: Validation of Elevated Peroxiredoxin 1 in Patient Peripheral Circulation 
PLoS ONE  2011;6(9):e25056.
Background
Epithelial ovarian cancer (EOC) is the deadliest gynecologic malignancy in the United States. Unfortunately, a validated protein biomarker-screening test to detect early stage disease from peripheral blood has not yet been developed. The present investigation assesses the ability to identify tumor relevant proteins from ovarian cancer proximal fluids, including tissue interstitial fluid (TIF) and corresponding ascites, from patients with papillary serous EOC and translates these findings to targeted blood-based immunoassays.
Methodology/Principal Findings
Paired TIF and ascites collected from four papillary serous EOC patients at the time of surgery underwent immunodepletion, resolution by 1D gel electrophoresis and in-gel digestion for analysis by liquid chromatography-tandem mass spectrometry, which resulted in an aggregate identification of 569 and 171 proteins from TIF and ascites, respectively. Of these, peroxiredoxin I (PRDX1) was selected for validation in serum by ELISA and demonstrated to be present and significantly elevated (p = 0.0188) in 20 EOC patients with a mean level of 26.0 ng/mL (±9.27 SEM) as compared to 4.19 ng/mL (±2.58 SEM) from 16 patients with normal/benign ovarian pathology.
Conclusions/Significance
We have utilized a workflow for harvesting EOC-relevant proximal biofluids, including TIF and ascites, for proteomic analysis. Among the differentially abundant proteins identified from these proximal fluids, PRDX1 was demonstrated to be present in serum and shown by ELISA to be elevated by nearly 6-fold in papillary serous EOC patients relative to normal/benign patients. Our findings demonstrate the facile ability to discover potential EOC-relevant proteins in proximal fluids and confirm their presence in peripheral blood serum. In addition, our finding of elevated levels of PRDX1 in the serum of EOC patients versus normal/benign patients warrants further evaluation as a tumor specific biomarker for EOC.
doi:10.1371/journal.pone.0025056
PMCID: PMC3184097  PMID: 21980378
13.  Preformulation and stability in biological fluids of the retrocyclin RC-101, a potential anti-HIV topical microbicide 
Background
RC-101, a cationic peptide retrocyclin analog, has in vitro activity against HIV-1. Peptide drugs are commonly prone to conformational changes, oxidation and hydrolysis when exposed to excipients in a formulation or biological fluids in the body, this can affect product efficacy. We aimed to investigate RC-101 stability under several conditions including the presence of human vaginal fluids (HVF), enabling the efficient design of a safe and effective microbicide product. Stability studies (temperature, pH, and oxidation) were performed by HPLC, Circular Dichroism, and Mass Spectrometry (LC-MS/MS). Additionally, the effect of HVF on formulated RC-101 was evaluated with fluids collected from healthy volunteers, or from subjects with bacterial vaginosis (BV). RC-101 was monitored by LC-MS/MS for up to 72 h.
Results
RC-101 was stable at pH 3, 4, and 7, at 25 and 37°C. High concentrations of hydrogen peroxide resulted in less than 10% RC-101 reduction over 24 h. RC-101 was detected 48 h after incubation with normal HVF; however, not following incubation with HVF from BV subjects.
Conclusions
Our results emphasize the importance of preformulation evaluations and highlight the impact of HVF on microbicide product stability and efficacy. RC-101 was stable in normal HVF for at least 48 h, indicating that it is a promising candidate for microbicide product development. However, RC-101 stability appears compromised in individuals with BV, requiring more advanced formulation strategies for stabilization in this environment.
doi:10.1186/1742-6405-8-27
PMCID: PMC3199744  PMID: 21801426
14.  Identifier mapping performance for integrating transcriptomics and proteomics experimental results 
BMC Bioinformatics  2011;12:213.
Background
Studies integrating transcriptomic data with proteomic data can illuminate the proteome more clearly than either separately. Integromic studies can deepen understanding of the dynamic complex regulatory relationship between the transcriptome and the proteome. Integrating these data dictates a reliable mapping between the identifier nomenclature resultant from the two high-throughput platforms. However, this kind of analysis is well known to be hampered by lack of standardization of identifier nomenclature among proteins, genes, and microarray probe sets. Therefore data integration may also play a role in critiquing the fallible gene identifications that both platforms emit.
Results
We compared three freely available internet-based identifier mapping resources for mapping UniProt accessions (ACCs) to Affymetrix probesets identifications (IDs): DAVID, EnVision, and NetAffx. Liquid chromatography-tandem mass spectrometry analyses of 91 endometrial cancer and 7 noncancer samples generated 11,879 distinct ACCs. For each ACC, we compared the retrieval sets of probeset IDs from each mapping resource. We confirmed a high level of discrepancy among the mapping resources. On the same samples, mRNA expression was available. Therefore, to evaluate the quality of each ACC-to-probeset match, we calculated proteome-transcriptome correlations, and compared the resources presuming that better mapping of identifiers should generate a higher proportion of mapped pairs with strong inter-platform correlations. A mixture model for the correlations fitted well and supported regression analysis, providing a window into the performance of the mapping resources. The resources have added and dropped matches over two years, but their overall performance has not changed.
Conclusions
The methods presented here serve to achieve concrete context-specific insight, to support well-informed decisions in choosing an ID mapping strategy for "omic" data merging.
doi:10.1186/1471-2105-12-213
PMCID: PMC3124437  PMID: 21619611
15.  A Sulfilimine Bond Identified in Collagen IV 
Science (New York, N.Y.)  2009;325(5945):1230-1234.
Collagen IV networks are ancient proteins of basement membranes that underlie epithelia in metazoa from sponge to human. The networks provide structural integrity to tissues and serve as ligands for integrin cell-surface receptors. They are assembled by oligomerization of triple-helical protomers and are covalently cross-linked, a key reinforcement that stabilizes networks. We used Fourier-transform ion cyclotron resonance mass spectrometry and nuclear magnetic resonance spectroscopy to show that a sulfilimine bond (-S=N-) crosslinks hydroxylysine-211 and methionine-93 of adjoining protomers, a bond not previously found in biomolecules. This bond, the nitrogen analog of a sulfoxide, appears to have arisen at the divergence of sponge and cnidaria, an adaptation of the extracellular matrix in response to mechanical stress in metazoan evolution.
doi:10.1126/science.1176811
PMCID: PMC2876822  PMID: 19729652
16.  Fatty Acid Synthase Is Upregulated during HCV Infection and Regulates HCV Entry and Production§ 
Hepatology (Baltimore, Md.)  2008;48(5):1396-1403.
Hepatitis C virus (HCV) is a major human pathogen that causes serious illness including acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Using a mass spectrometry-based proteomics approach, we have identified 175 proteins from a cell culture supernatant fraction containing HCV JFH1 virus, among which fatty acid synthase (FASN), the multifunctional enzyme catalyzing the de novo synthesis of fatty acids, was confirmed to be highly enriched. Subsequent studies showed that FASN expression increased in the human hepatoma cell line, Huh7 or its derivative, upon HCV infection. Blocking FASN activity by a pharmacological inhibitor C75 led to decreased HCV production. Reduction of FASN by RNA interference (RNAi) suppressed viral replication in both replicon and infection systems. Remarkably, FASN appeared to be selectively required for the expression of claudin-1 (CLDN1), a tight junction (TJ) protein that was recently identified as an entry co-receptor for HCV (1), but not for the expression of another HCV co-receptor, CD81. The decrease in CLDN1 expression resulting from FASN inhibition was accompanied by a decrease in transepithelial electric resistance (TER) of Huh7 cells, implying a reduction in the relative tightness of the cell monolayer. Consequently, the entry of HIV-HCV pseudotypes (HCVpp) was significantly inhibited in C75 treated Huh7 cells. Conclusion: As far as we know, this is the first line of evidence that demonstrates that HCV infection directly induces FASN expression, and thus suggests a possible mechanism by which HCV infection alters the cellular lipid profile and causes diseases such as steatosis.
doi:10.1002/hep.22508
PMCID: PMC2614928  PMID: 18830996
Hepatitis C virus; fatty acid synthase; viral replication; viral entry; Claudin-1
17.  Oxidative Inactivation of Key Mitochondrial Proteins Leads to Dysfunction and Injury in Hepatic Ischemia Reperfusion 
Gastroenterology  2008;135(4):1344-1357.
Summary
Background & Aims
Ischemia-reperfusion (I/R) is a major mechanism of liver injury following hepatic surgery or transplantation. Despite numerous reports on the role of oxidative/nitrosative stress and mitochondrial dysfunction in hepatic I/R injury, the proteins that are oxidatively-modified during I/R damage are poorly characterized. This study was aimed at investigating the oxidatively-modified proteins underlying the mechanism for mitochondrial dysfunction in hepatic I/R injury. We also studied the effects of a superoxide dismutase mimetic/peroxynitrite scavenger metalloporphyrin MnTMPyP on oxidatively-modified proteins and their functions.
Methods
The oxidized and/or S-nitrosylated mitochondrial proteins from I/R-injured mouse livers with or without MnTMPyP pretreatment, were labeled with biotin-N-maleimide, purified with streptavidin-agarose and resolved by two-dimensional gel electrophoresis. The identities of the oxidatively-modified proteins were determined using mass-spectrometric analysis. Liver histopathology, serum transaminase levels, nitrosative stress markers and the activities of oxidatively-modified mitochondrial proteins were measured.
Results
Comparative two-dimensional gel analysis revealed markedly increased numbers of oxidized and S-nitrosylated mitochondrial proteins following hepatic I/R injury. Many key mitochondrial enzymes involved in cellular defense, fat metabolism, energy supply, and chaperones were identified as being oxidatively-modified proteins. Pretreatment with MnTMPyP attenuated the I/R-induced increased serum transaminase levels, histological damage, increased iNOS expression, and S-nitrosylation and/or nitration of various key mitochondrial proteins. MnTMPyP pretreatment also restored I/R-induced suppressed activities of mitochondrial aldehyde dehydrogenase, 3-ketoacyl-CoA thiolases, and ATP synthase.
Conclusions
These results suggest that increased nitrosative stress is critically important in promoting S-nitrosylation and nitration of various mitochondrial proteins, leading to mitochondrial dysfunction with decreased energy supply and hepatic injury.
doi:10.1053/j.gastro.2008.06.048
PMCID: PMC2597302  PMID: 18778711
Ischemia-reperfusion injury; liver mitochondria dysfunction; redox biology; Cysteine oxidation; S-nitrosylation; metalloporphyrin peroxynitrite scavenger
18.  Application of a Global Proteomic Approach to Archival Precursor Lesions: Deleted in Malignant Brain Tumors 1 and Tissue Transglutaminase 2 Are Upregulated in Pancreatic Cancer Precursors 
Background
Pancreatic cancer is an almost uniformly fatal disease, and early detection is a critical determinant of improved survival. A variety of noninvasive precursor lesions of pancreatic adenocarcinoma have been identified, which provide a unique opportunity for intervention prior to onset of invasive cancer. Biomarker discovery in precursor lesions has been hampered by the ready availability of fresh specimens, and limited yields of proteins suitable for large scale screening.
Methods
We utilized Liquid Tissue®, a novel technique for protein extraction from archival formalin-fixed material, and mass spectrometry to conduct a global proteomic analysis of an intraductal papillary mucinous neoplasm (IPMN). Tissue microarrays comprised of 38 IPMNs were used for validation of candidate proteins.
Results
The proteomic analysis of the IPMN Liquid Tissue lysate resulted in identification of 1,534 peptides corresponding to 523 unique proteins. A subset of 25 proteins was identified that had previously been reported as upregulated in pancreatic cancer. Immunohistochemical analysis for two of these, deleted in malignant brain tumors 1 (DMBT1) and tissue transglutaminase 2 (TGM2), confirmed their overexpression in IPMNs.
Conclusion
Global proteomics analysis using the Liquid Tissue workflow is a feasible approach for unbiased biomarker discovery in limited archival material, particularly applicable to precursor lesions of cancer.
doi:10.1159/000161012
PMCID: PMC2711211  PMID: 18849643
Pancreatic cancer; Intraductal papillary mucinous neoplasms; Proteomics; Mass spectrometry
19.  The heavy metal cadmium induces valosin-containing protein (VCP)-mediated aggresome formation 
Toxicology and applied pharmacology  2008;228(3):351-363.
Cadmium (Cd2+) is a heavy metal ion known to have a long biological half-life in humans. Accumulating evidence shows that exposure to Cd2+ is associated with neurodegenerative diseases characterized by the retention of ubiquitinated and misfolded proteins in the lesions. Here, we report that Cd2+ directly induces the formation of protein inclusion bodies in cells. The protein inclusion body is an aggresome, a major organelle for collecting ubiquitinated or misfolded proteins. Our results show that aggresomes are enriched in the detergent-insoluble fraction of Cd2+-treated cell lysates. Proteomic analysis identified 145 proteins in the aggresome-enriched fractions. One of the proteins is the highly conserved valosin-containing protein (VCP), which has been shown to colocalize with aggresomes and bind ubiquitinated proteins through its N domain (#1–200). Our subsequent examination of VCP's role in the formation of aggresomes induced by Cd2+ indicate that the C-terminal tail (#780-806) of VCP interacts with histone deacetylase HDAC6, a mediator for aggresome formation, suggesting that VCP participates in transporting ubiquitinated proteins to aggresomes. This function of VCP is impaired by inhibition of the deacetylase activity of HDAC6 or by over-expression of VCP mutants that do not bind ubiquitinated proteins or HDAC6. Our results indicate that Cd2+ induces the formation of protein inclusion bodies by promoting the accumulation of ubiquitinated proteins in aggresomes through VCP and HDAC6. Our delineation of the role of VCP in regulating cell responses to ubiquitinated proteins has important implications for understanding Cd2+ toxicity and associated diseases.
doi:10.1016/j.taap.2007.12.026
PMCID: PMC2692476  PMID: 18261755
Cadmium; Aggresome; Ubiquitin proteasome system; HDAC6
20.  Mass Spectrometry Reveals Specific and Global Molecular Transformations during Viral Infection 
Journal of proteome research  2006;5(9):2405-2416.
Mass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Rock House Virus (FHV) proteins in response to FHV infection of Drosophila cells were monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins were identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up regulation of the Drosophila apoptotic croquemort protein, and the down regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.
doi:10.1021/pr060215t
PMCID: PMC2566936  PMID: 16944953
virus; protein regulation; viral infection; metabolites; isotope labeling; mass spectrometry
21.  Application of a Global Proteomic Approach to Archival Precursor Lesions: Deleted in Malignant Brain Tumors 1 and Tissue Transglutaminase 2 Are Upregulated in Pancreatic Cancer Precursors 
Pancreatology  2008;8(6):608-616.
Background
Pancreatic cancer is an almost uniformly fatal disease, and early detection is a critical determinant of improved survival. A variety of noninvasive precursor lesions of pancreatic adenocarcinoma have been identified, which provide a unique opportunity for intervention prior to onset of invasive cancer. Biomarker discovery in precursor lesions has been hampered by the ready availability of fresh specimens, and limited yields of proteins suitable for large scale screening.
Methods
We utilized Liquid Tissue®, a novel technique for protein extraction from archival formalin-fixed material, and mass spectrometry to conduct a global proteomic analysis of an intraductal papillary mucinous neoplasm (IPMN). Tissue microarrays comprised of 38 IPMNs were used for validation of candidate proteins.
Results
The proteomic analysis of the IPMN Liquid Tissue lysate resulted in identification of 1,534 peptides corresponding to 523 unique proteins. A subset of 25 proteins was identified that had previously been reported as upregulated in pancreatic cancer. Immunohistochemical analysis for two of these, deleted in malignant brain tumors 1 (DMBT1) and tissue transglutaminase 2 (TGM2), confirmed their overexpression in IPMNs.
Conclusion
Global proteomics analysis using the Liquid Tissue workflow is a feasible approach for unbiased biomarker discovery in limited archival material, particularly applicable to precursor lesions of cancer.
doi:10.1159/000161012
PMCID: PMC2711211  PMID: 18849643
Pancreatic cancer; Intraductal papillary mucinous neoplasms; Proteomics; Mass spectrometry
22.  Increased serum levels of complement C3a anaphylatoxin indicate the presence of colorectal tumors 
Gastroenterology  2006;131(4):1020-1284.
Background & Aims
Late diagnosis of colorectal carcinomas results in a significant reduction of average survival times. Yet, despite screening programs about 70% of tumors are detected at advanced stages (UICC III/IV). We explored whether detection of malignant disease would be possible through identification of tumor specific protein biomarkers in serum samples.
Methods
A discovery set of sera from patients with colorectal malignancy (n=58) and healthy control individuals (n=32) were screened for potential differences using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Candidate proteins were identified, and their expression levels validated in independent sample sets using a specific immunoassay (ELISA).
Results
Utilizing class comparison and custom developed algorithms we identified several m/z values that were differentially expressed between the malignant samples and the healthy controls of the discovery set. Characterization of the most prominent m/z values revealed a member of the complement system, the stable form of C3a anaphylatoxin, i.e., C3a-desArg. Based on a specific ELISA, serum levels of complement C3a-desArg predicted the presence of colorectal malignancy in a blinded validation set (n=59) with a sensitivity of 96.8% and a specificity of 96.2%. Increased serum levels were also detected in 86.1% of independently collected sera from patients with colorectal adenomas (n=36), while only 5.6% were classified as normal.
Conclusion
Complement C3a-desArg is present at significantly higher levels in serum from patients with colorectal adenomas (p<0.0001) and carcinomas (p<0.0001) than in healthy individuals. This suggests that quantification of C3a-desArg levels could ameliorate existing screening tests for colorectal cancer.
doi:10.1053/j.gastro.2006.07.011
PMCID: PMC2532535  PMID: 17030172
Colorectal Cancer; Polyps; Screening; Serum; SELDI-TOF MS; C3a-desArg
23.  NMDA Di-Heteromeric Receptor Populations and Associated Proteins in Rat Hippocampus 
Subunit composition of NMDA receptors (NMDARs) determines a range of physiological properties, downstream signaling effects, and binding partners. Differential localization of NR2A- or NR2B-containing NMDARs within the neuron and subunit-specific protein associations may explain differences in NR2A and NR2B contributions to synaptic plasticity and excitotoxic cell death. This question is complicated by the existence of tri-heteromeric complexes (NR1/NR2A/NR2B). To date, no quantitative biochemical determinations have been made of the relative abundance of different NMDAR populations in intact hippocampus, the region extensively correlated with NMDAR-dependent long-term potentiation. We investigated subunit composition and subunit-specific interactions in CA1/CA2 of rat hippocampus. Using sequential immunoprecipitations to deplete either NR2A or NR2B, di-heteromeric NR1/NR2A and NR1/NR2B receptor populations were isolated from postnatal day (P)7 hippocampus and P42 and 6-month old CA1/CA2. Quantitative Western blot analysis revealed that 60–70% of NR2A and 70–85% of NR2B subunits were associated in NR1/NR2A or NR1/NR2B di-heteromeric complexes. Isolated di-heteromeric receptor fractions were used to examine NR2A- or NR2B-specific interactions with synapse-associated proteins. Our results indicate that NR2A- or NR2B-containing NMDARs associate similarly with PSD-95, SAP102, and PSD-93 at P42. However, NR2A-containing receptors co-immunoprecipitated a greater proportion of the synaptic proteins nNOS, Homer, and β-catenin. Finally, mass spectrometry analysis of isolated di-heteromeric receptors identified a novel NMDAR interactor, Collapsin Response Mediator Protein 2 (CRMP2), which preferentially associates with NR2B-containing di-heteromeric NMDARs. In summary, in rat hippocampus, NR2A and NR2B exist largely in di-heteromeric complexes that interact similarly with PSD-95-related proteins but are associated with different protein complexes.
doi:10.1523/JNEUROSCI.2155-07.2007
PMCID: PMC2263005  PMID: 17670980
Tri-heteromeric; MAGUK; PSD-95; CRMP2; Detergent solubility; Postsynaptic
24.  Parallel analysis of transcript and translation profiles: identification of metastasis-related signal pathways differentially regulated by drug and genetic modifications 
Journal of proteome research  2006;5(7):1555-1567.
Tumor metastasis is a complex multi-step process normally involving dysregulation of multiple signal transduction pathways. In this study we developed a novel approach to efficiently define dysreguated pathways associated with metastasis by comparing global gene and protein expressions of two distinct metastasis-suppressed models. Consequently, we identified common features shared by the two models which are potentially associated with metastasis.
The efficiency of metastasis from the highly aggressive polyoma middle T-induced mouse mammary tumors was suppressed by either prolonged caffeine exposure or by breeding the animal to a low metastastic mouse strain. Molecular profiles of the primary tumors from both metastasis-suppressed classes were then derived to identify molecules and pathways that might underlie a common mechanism of metastasis. A number of differentially regulated genes and proteins were identified, including genes encoding basement membrane components, which were inversely related to metastatic efficiency. In addition, the analysis revealed that the Stat signal transduction pathways were potentially associated with metastasis inhibition, as demonstrated by enhanced Stat1 activation, and decreased Stat5 phosphorylation in both genetic and pharmacological modification models. Tumor cells of low-metastatic genotypes also demonstrated anti-apoptotic properties. The common changes of these pathways in all of the metastasis-suppressed systems suggest that they may be critical components in the metastatic cascade, at least in this model system. Our data demonstrate that analysis of common changes in genes and proteins in a metastatic-related context greatly decrease the complexity of data analysis, and may serve as a screening tool to identify biological important factors from large scale data.
doi:10.1021/pr0504283
PMCID: PMC1501083  PMID: 16823962
systems biology; biological association network; metastasis; signaling pathway; microarray; isotope-coded affinity tag
25.  Increased oxidation and degradation of cytosolic proteins in alcohol-exposed mouse liver and hepatoma cells 
Proteomics  2006;6(4):1250-1260.
We recently developed a sensitive method using biotin-N-maleimide (biotin-NM) as a probe to positively identify oxidized mitochondrial proteins. In this study, biotin-NM was used to identify oxidized cytosolic proteins in alcohol-fed mouse livers. Alcohol treatment for 6 weeks elevated the levels of CYP2E1 and nitrotyrosine, a marker of oxidative stress. Markedly increased levels of oxidized proteins were detected in alcohol-fed mouse livers compared to pair-fed controls. The biotin-NM-labeled oxidized proteins from alcohol-exposed mouse livers were subsequently purified with streptavidin-agarose and resolved on 2-DE. More than 90 silver-stained protein spots that displayed differential intensities on 2-D gels were identified by mass spectrometry. Peptide sequence analysis revealed that many enzymes or proteins involved in stress response, chaperone activity, intermediary metabolism, and antioxidant defense systems such as peroxiredoxin were oxidized after alcohol treatment. Smaller fragments of many proteins were repeatedly detected only in alcohol-fed mice, indicating that many oxidized proteins after alcohol exposure were degraded. Immunoblot results showed that the level of oxidized peroxiredoxin (inactivated) was markedly increased in the alcohol-exposed mouse livers and ethanol-sensitive hepatoma cells compared to the corresponding controls. Our results may explain the underlying mechanism for cellular dysfunction and increased susceptibility to other toxic agents following alcohol-mediated oxidative stress.
doi:10.1002/pmic.200500447
PMCID: PMC1368983  PMID: 16408314
Alcoholism; CYP2E1; Oxidative stress; Protein Oxidation; Protein Degradation; Peroxiredoxin; Biotin-NM, biotin-N-maleimide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose regulated protein 78 kDa; FTHF-DH, formyltetrahydrofolate dehydrogenase; HRP, horse radish peroxidase; HSP, heat shock protein; iNOS, inducible nitric oxide synthase; MAb, monoclonal antibody; MAT: methionine S-adenosyltransferase; NEM, N-ethylmaleimide; PDI, protein disulfide isomerase; Prx, peroxiredoxin; Prx- SO3, oxidized-Prx; SAHH,S-adenosyl homocysteine hydrolase

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