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1.  Differential expression of proteins in naïve and IL-2 stimulated primary human NK cells identified by global proteomic analysis 
Journal of proteomics  2013;91:151-163.
Natural killer (NK) cells efficiently cytolyse tumors and virally infected cells. Despite the important role that interleukin (IL)-2 plays in stimulating the proliferation of NK cells and increasing NK cell activity, little is known about the alterations in the global NK cell proteome following IL-2 activation. To compare the proteomes of naïve and IL-2-activated primary NK cells and identify key cellular pathways involved in IL-2 signaling, we isolated proteins from naïve and IL-2-activated NK cells from healthy donors, the proteins were trypsinized and the resulting peptides were analyzed by 2D LC ESI-MS/MS followed by label-free quantification. In total, more than 2000 proteins were identified from naïve and IL-2-activated NK cells where 383 proteins were found to be differentially expressed following IL-2 activation. Functional annotation of IL-2 regulated proteins revealed potential targets for future investigation of IL-2 signaling in human primary NK cells. A pathway analysis was performed and revealed several pathways that were not previously known to be involved in IL-2 response, including ubiquitin proteasome pathway, integrin signaling pathway, platelet derived growth factor (PDGF) signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway and Wnt signaling pathway.
doi:10.1016/j.jprot.2013.06.024
PMCID: PMC4326111  PMID: 23806757
NK cells; IL-2 signaling; Mass spectrometry pathways; Proteomics; Lab-free quantification; Multi-dimensional separation
2.  [No title available] 
PMCID: PMC3745587  PMID: 23583696
3.  [No title available] 
PMCID: PMC3806883  PMID: 23702328
4.  [No title available] 
PMCID: PMC3951211  PMID: 23811049
5.  Using a Cell Line Breast Cancer Progression System to Identify Biomarker Candidates 
Journal of proteomics  2013;96:173-183.
Secreted and plasma membrane glycoproteins are considered excellent candidates for disease biomarkers. Herein we describe the identification of secreted and plasma membrane glycoproteins that are differentially expressed among a family of three breast cancer cell lines that models the progression of breast cancer. Using two-dimensional liquid chromatography-tandem mass spectrometry we identified more than 40 glycoproteins that were differentially expressed in either the premalignant (MCF10AT) or the fully malignant (MCF10CA1a) cell lines of this model system. Comparative analysis revealed that the differentially expressed breast cancer progression-associated glycoproteins were among the most highly expressed in the malignant (MCF10CA1a) breast cancer cell line; a subset of these were detected only in the malignant line; and others were detected in the malignant line at levels 25 to 50 times greater than in the benign (MCF10A) line. Using the results from this model cell system as a guide, we then carried out glycoproteomic analyses of normal and cancerous breast tissue lysates. Eleven of the glycoproteins differentially expressed in the breast cell lines were identified in the tissue lysates. Among these glycoproteins, collagen alpha-1 (XII) chain was expressed at dramatically higher (∼10-fold) levels in breast cancer than in normal tissue.
doi:10.1016/j.jprot.2013.11.006
PMCID: PMC3920745  PMID: 24262153
Breast cancer; glycoproteomics; differential expression; non-malignant vs. malignant; label-free quantitation; spectral counts
6.  Expression of the mevalonate pathway enzymes in the Lutzomyia longipalpis (Diptera: Psychodidae) sex pheromone gland demonstrated by an integrated proteomic approach 
Journal of proteomics  2013;96:117-132.
In Latin America, Lutzomyia longipalpis is the main vector of the protozoan parasite Leishmania infantum, which is the causal agent of American Visceral Leishmaniasis. This insect uses male-produced pheromones for mate recognition. Elucidation of pheromone biogenesis or its regulation may enable molecular strategies for mating disruption and, consequently, the vector's population management. Motivated by our recent results of the transcriptomic characterization of the L. longipalpis pheromone gland, we performed a proteomic analysis of this tissue combining SDS-PAGE, and mass spectrometry followed by an integrative data analysis. Considering that annotated genome sequences of this sand fly are not available, we designed an alternative workflow searching MS/MS data against two customized databases using three search engines: Mascot, OMSSA and ProLuCID. A total of 542 proteins were confidently characterized, 445 of them using a Uniref100-insect protein database, and 97 using a transcript translated database. In addition, use of PEAKS for de novo peptide sequencing of MS/MS data confirmed ∼90% identifications made with the combination of the three search engines. Our results include the identification of six of the seven enzymes of the mevalonate-pathway, plus the enzymes involved in sesquiterpenoid biosynthesis, all of which are proposed to be involved in pheromone production in L. longipalpis.
Biological significance
L. longipalpis is the main vector of the protozoan parasite L. infantum, which is the causal agent of American Visceral Leishmaniasis. One of the control measures of such disease is focused on vector population control. As this insect uses male-produced pheromones for mate recognition, the elucidation of pheromone biogenesis or its regulating process may enable molecular strategies for mating disruption and, consequently, this vector's population management. On this regard, in this manuscript we report expression evidence, at the protein level, of several molecules potentially involved in the pheromone production of L. longipalpis. Our results include the identification of the mevalonate-pathway enzymes, plus the enzymes involved in sesquiterpenoid biosynthesis, all of which are proposed to be involved in pheromone production in L. longipalpis. In addition, considering that the annotated genome sequences of this sand fly are not yet available, we designed an alternative workflow searching MS/MS data against proteomic and transcript translated customized databases, using three search engines: Mascot, OMSSA, and ProLuCID. In addition, a de novo peptide sequencing software (PEAKS) was used to further analyze the MS/MS data. This approach made it possible to identify and annotate 542 proteins for the pheromone gland of L. longipalpis. Importantly, all annotated protein sequences and raw data are available for the research community in protein repositories that provide free access to the data.
doi:10.1016/j.jprot.2013.10.028
PMCID: PMC3917562  PMID: 24185139
Lutzomyia longipalpis; Male pheromone gland; Proteomics; Proteome; Mevalonate pathway
7.  Discovery of sialyl Lewis A and Lewis X modified protein cancer biomarkers using high density antibody arrays 
Journal of proteomics  2013;96:291-299.
We report on a high-dimensional method to globally profile glycoproteins that are modified with sialyl Lewis A or Lewis X glycans. Specifically, glycoproteins in serum or plasma are fractionated on a high-density antibody microarray (i.e., each are localized to their specific antibody spot) and are specifically detected via fluorescently labeled anti-sialyl Lewis A or anti- Lewis X antibodies with quantification in a microarray scanner. Non-glycosylated proteins or glycoproteins with other glycan motifs do not interfere with this assay. The whole process is very rapid and applicable for high-throughput screening without the need for purification of glycoproteins from the samples. Using these methods, sialyl Lewis A or Lewis X moieties were found to be expressed on many previously unreported secreted or membrane associated proteins. Furthermore, the combination of sialyl Lewis A or Lewis X content with protein level increased the ability of certain glycoproteins to distinguish 30 patients with stage III and IV colon cancer from 60 control samples. Thus, this highly sensitive method is capable of discovering novel specific glycan modifications on proteins, many of which will likely be useful for disease detection and monitoring.
doi:10.1016/j.jprot.2013.10.030
PMCID: PMC3946870  PMID: 24185138
glycoproteins; glycans; sialyl Lewis A; Lewis X; cancer biomarker; antibody array
8.  Proteome of the porosome complex in human airway epithelia: Interaction with the cystic fibrosis transmembrane conductance regulator (CFTR) 
Journal of proteomics  2013;96:82-91.
The surface of the airways is coated with a thin film of mucus composed primarily of mucin, which is under continuous motion via ciliary action. Mucin not only serves to lubricate the airways epithelia, but also functions as a trap for foreign particles and pathogens, thereby assisting in keeping the airways clean and free of particulate matter and infections. Altered mucin secretion especially increased mucin viscosity, results in mucin stagnation due to the inability of the cilia to propel them, leading to infections and diseases such as cystic fibrosis (CF). Since porosomes have been demonstrated to be the secretory portals at the cell plasma membrane in cells, their presence, structure, and composition in the mucin-secreting human airway epithelial cell line Calu-3 expressing CF transmembrane receptor (CFTR), were investigated. Atomic force microscopy (AFM) of Calu-3 cells demonstrates the presence of approximately 100 nm in diameter porosome openings at the plasma membrane surface. Electron microscopy confirms the AFM results, and tandem mass spectrometry and immunoanalysis performed on isolated Calu-3 porosomes, reveal the association of CFTR with the porosome complex. These new findings will facilitate understanding of CFTR–porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease.
Biological significance
In the present study, the porosome proteome in human airway epithelia has been determined. The interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the porosome complex in the human airway epithelia is further demonstrated. The possible regulation by CFTR on the quality of mucus secretion via the porosome complex at the cell plasma membrane is hypothesized. These new findings will facilitate understanding of CFTR–porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease.
doi:10.1016/j.jprot.2013.10.041
PMCID: PMC3961139  PMID: 24220302
Human airway epithelia porosome; proteome; Porosome–CFTR interaction; Mucus secretion; Tandem mass spectrometry
9.  [No title available] 
PMCID: PMC4294458  PMID: 24211403
10.  Combined enrichment of neuromelanin granules and synaptosomes from human substantia nigra pars compacta tissue for proteomic analysis 
Journal of proteomics  2013;94:202-206.
This article gives a detailed description of a protocol using density gradient centrifugation for the enrichment of neuromelanin granules and synaptosomes from low amounts (≥0.15 g) of human substantia nigra pars compacta tissue. This has a great advantage compared to already existing methods as it allows for the first time (i) a combined enrichment of neuromelanin granules and synaptosomes and (ii) just minimal amounts of tissue necessary to enable donor specific analysis. Individual specimens were classified as control or diseased according to clinical evaluation and neuropathological examination. For the enrichment of synaptosomes and neuromelanin granules from the same tissue sample density gradient centrifugations using Percoll® and Iodixanol were performed. The purity of resulting fractions was checked by transmission electron microscopy. We were able to establish a reproducible and easy to handle protocol combining two different density gradient centrifugations: using an Iodixanol gradient neuromelanin granules were enriched and in parallel, from the same sample, a fraction of synaptosomes with high purity using a Percoll® gradient was obtained. Our subfractionation strategy will enable a subsequent in depth proteomic characterization of neurodegenerative processes in the substantia nigra pars compacta in patients with Parkinson’s disease and dementia with Lewy bodies compared to appropriate controls.
doi:10.1016/j.jprot.2013.07.015
PMCID: PMC4083096  PMID: 23917253
Neuromelanin; Synaptosomes; Density gradient; Substantia nigra pars compacta; Parkinson’s disease
11.  Manganese regulation of virulence factors and oxidative stress resistance in Neisseria gonorrhoeae 
Journal of proteomics  2009;73(5):899-916.
Neisseria gonorrhoeae has evolved a complex and novel network of oxidative stress responses, including defense mechanisms that are dependent on manganese (Mn). We performed systematic analyses at the transcriptomic and proteomic (1D SDS-PAGE and Isotope-Coded Affinity Tag [ICAT]) levels to investigate the global expression changes that take place in a high Mn environment, which results in a Mn-dependent oxidative stress resistance phenotype. These studies revealed that 97 proteins are regulated at the post-transcriptional level under conditions of increased Mn concentration, including proteins involved in virulence (eg. Pilin, a key adhesin), oxidative stress defence (eg. superoxide dismutase), cellular metabolism, protein synthesis, RNA processing and cell division. Mn regulation of inorganic pyrophosphatase (Ppa) indicated the potential involvement of phosphate metabolism in the Mn-dependent oxidative stress defense. A detailed analysis of the role of Ppa and polyphosphate kinase (Ppk) in the gonococcal oxidative stress response revealed that ppk and ppa mutant strains showed increased resistance to oxidative stress. Investigation of these mutants grown with high Mn suggests that phosphate and pyrophosphate are involved in Mn-dependent oxidative stress resistance.
doi:10.1016/j.jprot.2009.12.001
PMCID: PMC4238106  PMID: 20004262
manganese; microarray; Neisseria gonorrhoeae; oxidative stress; pili; proteomics
12.  Alternative cellular roles for proteins identified using proteomics 
Journal of proteomics  2012;75(13):4184-4185.
doi:10.1016/j.jprot.2012.04.049
PMCID: PMC4234028  PMID: 22579753
Adhesion complexes; Cell adhesion; Mitosis; Proteomics; Signalling
13.  Oxidative Stress and Protein Carbonylation in Adipose Tissue - Implications for Insulin Resistance and Diabetes Mellitus 
Journal of proteomics  2013;92:10.1016/j.jprot.2013.04.002.
While historically considered simply as a depot for excess energy, white adipose tissue is a dynamically active endocrine organ capable of responding to a variety of efferent stimuli resulting in the synthesis and secretion of peptides, proteins and metabolites that serve as signal transducers to the peripheral and central circulation. Such regulation controls a variety of physiological processes including energy expenditure, food intake, reproductive capacity and responsiveness to insulin. Indeed, accumulation of inflammatory cells in white adipose tissue is considered to be causative in the development of insulin resistance and eventually type 2 diabetes mellitus. A large body of evidence suggests that oxidative stress in adipose tissue not only correlates with insulin resistance but is also causative in its development. Moreover, using the available plasma oxidative stress biomarkers, many clinical studies have shown the presence of systemic oxidative stress in obese insulin resistant subjects, and its decrease after the successful treatment of obesity. In this review we emphasize the role of protein carbonylation in dysfunctional obese white adipose tissue and its metabolic implications. We focus on glutathione S-transferase A4 as the key enzyme for trans-4-hydroxy-2-nonenal and trans-4-oxo-2-nonenal removal from the cell, thus preventing protein carbonylation.
doi:10.1016/j.jprot.2013.04.002
PMCID: PMC3769464  PMID: 23584148
Oxidative Stress; Reactive Oxygen Species; Protein Carbonylation; Glutathione S-transferase; Adipose Tissue; Insulin Resistance
14.  Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: role of cysteine alkylation during sample processing 
Journal of proteomics  2013;92:299-312.
Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforms were GalNAc4Gal3 and GalNAc5Gal4 with one Gal-deficient site and GalNAc5Gal3 and GalNAc4Gal2 with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236.
doi:10.1016/j.jprot.2013.07.013
PMCID: PMC3984899  PMID: 23891555
human immunoglobulin A1 (IgA1); IgA nephropathy; O-glycosylation; glycopeptides; mass spectrometry; microgradient separation
15.  Post-translational Modifications of Pancreatic Fluid Proteins Collected via the Endoscopic Pancreatic Function Test (ePFT) 
Journal of proteomics  2013;92:10.1016/j.jprot.2013.01.033.
Background
Early diagnosis of chronic pancreatitis by mass spectrometry-based proteomics may result in therapies to retard or modify disease progression. We aimed to identify differences in posttranslational modifications (PTMs) in pancreatic fluid proteins from individuals with chronic pancreatitis (n=9) and non-pancreatitis controls (n=9).
Methods
We collected proteomic data from pancreatic fluid using mass spectrometry techniques. We performed database searches with emphasis on PTMs using ProteinPilot. We compared the frequency of specific PTMs in pancreatic fluid between cohorts and also to those identified in bile, gastroduodenal fluid, urine, and pancreatic duct and stellate cell lysates.
Results
We identified 97 PTMs in endoscopically-collected pancreatic fluid, of which 11 were identified exclusively in one cohort and 9 others were significantly different in frequency between cohorts. Comparing pancreatic fluid with other specimens revealed differences in specific PTM frequencies, indicating that the identified PTMs were not merely artifacts of sample processing.
Conclusions
We determined PTMs of proteins extracted from pancreatic fluid which differed in frequency in chronic pancreatitis patients verses controls. Such PTMs may serve as biomarker candidates of chronic pancreatitis upon validation with larger cohorts. The analysis of the PTM profile of pancreatic fluid proteins offers an alternative method to standard protein-based biomarker discovery.
doi:10.1016/j.jprot.2013.01.033
PMCID: PMC3731404  PMID: 23500127
pancreas; pancreas juice; pancreatic function test; biomarkers; chronic pancreatitis
16.  Site specific identification of endogenous S-nitrosocysteine proteomes 
Journal of proteomics  2013;92:10.1016/j.jprot.2013.05.033.
Cysteine S-nitrosylation is a post-translational modification regulating protein function and nitric oxide signaling. Herein the selectivity, reproducibility, and sensitivity of a mass spectrometry-based proteomic method for the identification of endogenous S-nitrosylated proteins is outlined. The method enriches for either S-nitrosylated proteins or peptides through covalent binding of the cysteine sulfur with phenylmercury at pH=6.0. Phenylmercury reacts selectively and efficiently with S-nitrosocysteine since no reactivity can be documented for disulfides, sulfinic or sulfonic acids, S-glutathionylated, S-alkylated or S- sulfhydrylated cysteine residues. A specificity of 97 ± 1 % for the identification of S-nitrosocysteine peptides in mouse liver tissue is achieved by the inclusion of negative controls. The method enables the detection of 36 S-nitrosocysteine peptides starting with 5 pmoles S-nitrosocysteine/mg of total tissue protein. Both the percentage of protein molecules modified as well as the occupancy by S-nitrosylation can be determined. Overall, selective, sensitive and reproducible enrichment of S-nitrosylated proteins and peptides is achieved by the use of phenylmercury. The inclusion of appropriate negative controls secures the precise identification of endogenous S-nitrosylated sites and proteins in biological samples.
doi:10.1016/j.jprot.2013.05.033
PMCID: PMC3818367  PMID: 23748021
cysteine modification; mass spectrometry; nitric oxide; protein S-nitrosylation
17.  Quantitative Phosphoproteomic Profiling of Human Non-Small Cell Lung Cancer Tumors 
Journal of proteomics  2013;91:10.1016/j.jprot.2013.07.023.
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Within the molecular scope of NCSLC, a complex landscape of dysregulated cellular signaling has emerged, defined largely by mutations in select mediators of signal transduction, including the epidermal growth factor receptor (EGFR) and anaplastic lymphoma (ALK) kinases. Consequently, these mutant kinases become constitutively activated and targets for chemotherapeutic intervention. Encouragingly, small molecule inhibitors of these pathways have shown promise in clinical trials or are approved for clinical use. However, many protein kinases are dysregulated in NSCLC without genetic mutations. To quantify differences in tumor cell signaling that are transparent to genomic methods, we established a super-SILAC internal standard derived from NSCLC cell lines grown in vitro and labeled with heavy lysine and arginine, and deployed them in a phosphoproteomics workflow. We identified 9019 and 8753 phosphorylation sites in two separate tumors. Relative quantification of phosphopeptide abundance between tumor samples allowed for the determination of specific hubs and pathways differing between each tumor. Sites downstream of Ras showed decreased inhibitory phosphorylation (Raf/Mek) and increased activating phosphorylation (Erk1/2) in one tumor versus another. In this way, we were able to quantitatively access oncogenic kinase signaling in primary human tumors.
doi:10.1016/j.jprot.2013.07.023
PMCID: PMC3825743  PMID: 23911959
phosphoproteomics; lung cancer; SILAC; quantitative proteomics
18.  Isolation and analysis of linker histones across cellular compartments 
Journal of proteomics  2013;91:10.1016/j.jprot.2013.08.022.
Analysis of histones, especially histone H1, is severely limited by immunological reagent availability. This paper describes the application of cellular fractionation with LC-MS for profiling histones in the cytosol and upon chromatin. First, we show that linker histones enriched by cellular fractionation gives less nuclear contamination and higher histone content than when prepared by nuclei isolation. Second, we profiled the soluble linker histones throughout the cell cycle revealing phosphorylation increases as cells reach mitosis. Finally, we monitored histone H1.2–H1.5 translocation to the cytosol in response to the CDK inhibitor flavopiridol in primary CLL cells treated ex vivo. Data shows all H1 variants translocate in response to drug treatment with no specific order to their cytosolic appearance. The results illustrate the utility of cellular fractionation in conjunction with LC-MS for the analysis of histone H1 throughout the cell.
doi:10.1016/j.jprot.2013.08.022
PMCID: PMC3863389  PMID: 24013129
Histone H1; LC-MS; Cellular Compartmentalization
19.  Global Proteomic Analysis in Trypanosomes Reveals Unique Proteins and Conserved Cellular Processes Impacted by Arginine Methylation 
Journal of proteomics  2013;91:210-225.
Arginine methylation is a common posttranslational modification with reported functions in transcription, RNA processing and translation, and DNA repair. Trypanosomes encode five protein arginine methyltransferases, suggesting that arginine methylation exerts widespread impacts on the biology of these organisms. Here, we performed a global proteomic analysis of T. brucei to identify arginine methylated proteins and their sites of modification. Using an approach entailing two-dimensional chromatographic separation, and alternating electron transfer dissociation and collision induced dissociation, we identified 1332 methylarginines in 676 proteins. The resulting data set represents the largest compilation of arginine methylated proteins in any organism to date. Functional classification revealed numerous arginine methylated proteins involved in flagellar function, RNA metabolism, DNA replication and repair, and intracellular protein trafficking. Thus, arginine methylation has the potential to impact aspects of T. brucei gene expression, cell biology, and pathogenesis. Interestingly, pathways with known methylated proteins in higher eukaryotes were identified in this study, but often different components of the pathway were methylated in trypanosomes. Methylarginines were often identified in glycine rich contexts, although exceptions to this rule were detected. Collectively, these data inform on a multitude of aspects of trypanosome biology and serve as a guide for the identification of homologous arginine methylated proteins in higher eukaryotes.
doi:10.1016/j.jprot.2013.07.010
PMCID: PMC3935770  PMID: 23872088
20.  A Multi-scale Strategy for Discovery of Novel Endogenous Neuropeptides in the Crustacean Nervous System 
Journal of proteomics  2013;91:10.1016/j.jprot.2013.06.021.
The conventional mass spectrometry (MS)-based strategy is often inadequate for the comprehensive characterization of various size neuropeptides without assistance of genomic information. This study evaluated sequence coverage of different size neuropeptides in two crustacean species, blue crab Callinectes sapidus and Jonah crab Cancer borealis using conventional MS methodologies and revealed limitations to mid- and large-size peptide analysis. Herein we attempt to establish a multi-scale strategy for simultaneous and confident sequence elucidation of various sizes of peptides in the crustacean nervous system. Nine novel neuropeptides spanning a wide range of molecular weights (0.9-8.2 kDa) were fully sequenced from a major neuroendocrine organ, the sinus gland of the spiny lobster Panulirus interruptus. These novel neuropeptides included seven allatostatin (A- and B-type) peptides, one crustacean hyperglycemic hormone precursor-related peptide, and one crustacean hyperglycemic hormone. Highly accurate multi-scale characterization of a collection of varied size neuropeptides was achieved by integrating traditional data-dependent tandem MS, improved bottom-up sequencing, multiple fragmentation technique-enabled top-down sequencing, chemical derivatization, and in silico homology search. Collectively, the ability to characterize a neuropeptidome with vastly differing molecule sizes from a neural tissue extract could find great utility in unraveling complex signaling peptide mixtures employed by other biological systems.
doi:10.1016/j.jprot.2013.06.021
PMCID: PMC3825776  PMID: 23806756
Neuropeptide; peptidomics; de novo sequencing; mass spectrometry; crustacean
21.  Proteomic analysis of human dental cementum and alveolar bone 
Journal of proteomics  2013;91:10.1016/j.jprot.2013.08.016.
Dental cementum (DC) is a bone-like tissue covering the tooth root and responsible for attaching the tooth to the alveolar bone (AB) via the periodontal ligament (PDL). Studies have unsuccessfully tried to identify factors specific to DC versus AB, in an effort to better understand DC development and regeneration. The present study aimed to use matched human DC and AB samples (n=7) to generate their proteomes for comparative analysis. Bone samples were harvested from tooth extraction sites, whereas DC samples were obtained from the apical root portion of extracted third molars. Samples were denatured, followed by protein extraction reduction, alkylation and digestion for analysis by nanoAcquity HPLC system and LTQ-FT Ultra. Data analysis demonstrated that a total of 318 proteins were identified in AB and DC. In addition to shared proteins between these tissues, 105 and 83 proteins exclusive to AB or DC were identified, respectively. This is the first report analyzing the proteomic composition of human DC matrix and identifying putative unique and enriched proteins in comparison to alveolar bone. These findings may provide novel insights into developmental differences between DC and AB, and identify candidate biomarkers that may lead to more efficient and predictable therapies for periodontal regeneration.
doi:10.1016/j.jprot.2013.08.016
PMCID: PMC3873800  PMID: 24007660
alveolar bone; dental cementum; proteomic analysis; periodontal ligament; dentin; superoxide dismutase 3
22.  Microparticle content of plasma for transfusion is influenced by the whole blood hold conditions: pre-analytical considerations for proteomic investigations 
Journal of proteomics  2012;76(0 0):211-219.
Microparticles (MPs) are shed from normal blood cells and may contribute to the coagulation potential of plasma. Transfusion of fresh frozen plasma (FFP) is used to correct coagulopathies and blood loss in trauma or major surgery. The role of MPs in FFP clinical efficacy is unknown. Regulations that govern the preparation of FFP vary in different countries. The aim of this study was to determine the effect of whole blood (WB)-hold conditions before FFP preparation on the MP profile. WB units were held at room temperature (RT) or combination of RT and refrigeration for up to 24hr before FFP preparation. The MP content in thawed FFP was measured to reflect transfusion practice. The absolute number of MPs in FFP increased with longer WB hold time. Refrigeration of WB may also promote increased generation of MPs. In particular the number of platelet-derived and phosphatidylserine-containing MPs, which are known to have procoagulant properties, increased. Lipid peroxidation increased with longer WB-hold time. Donor-related factors appear to govern lipid peroxidation levels. Holistic proteomic and coagulant analyses of FFP MPs is warranted. Such information could guide the choice of the optimal handling conditions of WB and the most relevant quality control procedures for FFP.
doi:10.1016/j.jprot.2012.07.013
PMCID: PMC4177036  PMID: 22813882
microparticles; phosphatidylserine; plasma; platelets; transfusion; whole blood
23.  Innovations in Proteomic Profiling of Cancers: Alternative Splice Variants as a New Class of Cancer Biomarker Candidates and Bridging of Proteomics with Structural Biology 
Journal of proteomics  2013;90:10.1016/j.jprot.2013.04.007.
Alternative splicing allows a single gene to generate multiple RNA transcripts which can be translated into functionally diverse protein isoforms. Current knowledge of splicing is derived mainly from RNA transcripts, with very little known about the expression level, 3D structures, and functional differences of the proteins. Splicing is a remarkable phenomenon of molecular and biological evolution. Studies which simply report up-regulation or down-regulation of protein or mRNA expression are confounded by the effects of mixtures of these isoforms. Besides understanding the net biological effects of the mixtures, we may be able to develop biomarker tests based on the observable differential expression of particular splice variants or combinations of splice variants in specific disease states. Here we review our work on differential expression of splice variant proteins in cancers and the feasibility of integrating proteomic analysis with structure-based conformational predictions of the differences between such isoforms.
doi:10.1016/j.jprot.2013.04.007
PMCID: PMC3841011  PMID: 23603631
splicing; differential expression; cancer biomarkers; computational modeling
24.  Identification and quantification of the basal and inducible Nrf2-dependent proteomes in mouse liver: Biochemical, pharmacological and toxicological implications 
Journal of Proteomics  2014;108(100):171-187.
The transcription factor Nrf2 is a master regulator of cellular defence: Nrf2 null mice (Nrf2(−/−)) are highly susceptible to chemically induced toxicities. We report a comparative iTRAQ-based study in Nrf2(−/−) mice treated with a potent inducer, methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-me; bardoxolone -methyl), to define both the Nrf2-dependent basal and inducible hepatoproteomes. One thousand five hundred twenty-one proteins were fully quantified (FDR < 1%). One hundred sixty-one were significantly different (P < 0.05) between WT and Nrf2(−/−) mice, confirming extensive constitutive regulation by Nrf2. Treatment with CDDO-me (3 mg/kg; i.p.) resulted in significantly altered expression of 43 proteins at 24 h in WT animals. Six proteins were regulated at both basal and inducible levels exhibiting the largest dynamic range of Nrf2 regulation: cytochrome P4502A5 (CYP2A5; 17.2-fold), glutathione-S-transferase-Mu 3 (GSTM3; 6.4-fold), glutathione-S-transferase Mu 1 (GSTM1; 5.9-fold), ectonucleoside-triphosphate diphosphohydrolase (ENTPD5; 4.6-fold), UDP-glucose-6-dehydrogenase (UDPGDH; 4.1-fold) and epoxide hydrolase (EPHX1; 3.0-fold). These proteins, or their products, thus provide a potential source of biomarkers for Nrf2 activity. ENTPD5 is of interest due to its emerging role in AKT signalling and, to our knowledge, this protein has not been previously shown to be Nrf2-dependent. Only two proteins altered by CDDO-me in WT animals were similarly affected in Nrf2(−/−) mice, demonstrating the high degree of selectivity of CDDO-me for the Nrf2:Keap1 signalling pathway.
Biological significance
The Nrf2:Keap1 signalling pathway is attracting considerable interest as a therapeutic target for different disease conditions. For example, CDDO-me (bardoxolone methyl) was investigated in clinical trials for the treatment of acute kidney disease, and dimethyl fumarate, recently approved for reducing relapse rate in multiple sclerosis, is a potent Nrf2 inducer. Such compounds have been suggested to act through multiple mechanisms; therefore, it is important to define the selectivity of Nrf2 inducers to assess the potential for off-target effects that may lead to adverse drug reactions, and to provide biomarkers with which to assess therapeutic efficacy. Whilst there is considerable information on the global action of such inducers at the mRNA level, this is the first study to catalogue the hepatic protein expression profile following acute exposure to CDDO-me in mice. At a dose shown to evoke maximal Nrf2 induction in the liver, CDDO-me appeared highly selective for known Nrf2-regulated proteins. Using the transgenic Nrf2(−/−) mouse model, it could be shown that 97% of proteins induced in wild type mice were associated with a functioning Nrf2 signalling pathway. This analysis allowed us to identify a panel of proteins that were regulated both basally and following Nrf2 induction. Identification of these proteins, which display a large magnitude of variation in their expression, provides a rich source of potential biomarkers for Nrf2 activity for use in experimental animals, and which may be translatable to man to define individual susceptibility to chemical stress, including that associated with drugs, and also to monitor the pharmacological response to Nrf2 inducers.
Graphical abstract
Highlights
•Liver proteomes from WT, Nrf2-null and Nrf2-induced mice were compared by iTRAQ•Of 1521 proteins quantified, 161 were regulated basally and 43 following induction•Six proteins were both basally and inducibly regulated, with high dynamic ranges•In order of fold change, these proteins were CYP2A5, GSTM3, GSTM1, ENTPD5, G6PD, EPHX1•These proteins may yield translatable biomarkers for clinical development
doi:10.1016/j.jprot.2014.05.007
PMCID: PMC4115266  PMID: 24859727
Nrf2; iTRAQ; ENTPD5; CYP2A5; Hepatoproteome; CDDO
25.  Protein isoform-specific validation defines multiple chloride intracellular channel and tropomyosin isoforms as serological biomarkers of ovarian cancer 
Journal of proteomics  2013;89:165-178.
New serological biomarkers for early detection and clinical management of ovarian cancer are urgently needed, and many candidates have been reported. A major challenge frequently encountered when validating candidates in patients is establishing quantitative assays that distinguish between highly homologous proteins. The current study tested whether multiple members of two recently discovered ovarian cancer biomarker protein families, chloride intracellular channel (CLIC) proteins and tropomyosins (TPM), were detectable in ovarian cancer patient sera. A multiplexed, label-free multiple reaction monitoring (MRM) assay was established to target peptides specific to all detected CLIC and TPM family members, and their serum levels were quantitated for ovarian cancer patients and non-cancer controls. In addition to CLIC1 and TPM1, which were the proteins initially discovered in a xenograft mouse model, CLIC4, TPM2, TPM3, and TPM4 were present in ovarian cancer patient sera at significantly elevated levels compared with controls. Some of the additional biomarkers identified in this homolog-centric verification and validation approach may be superior to the previously identified biomarkers at discriminating between ovarian cancer and non-cancer patients. This demonstrates the importance of considering all potential protein homologs and using quantitative assays for cancer biomarker validation with well-defined isoform specificity.
doi:10.1016/j.jprot.2013.06.016
PMCID: PMC3779132  PMID: 23792823

Results 1-25 (153)