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PubMed2005

Expression clustering reveals detailed co-expression patterns of functionally related proteins during B cell differentiation: a proteomic study using a combination of one-dimensional gel electrophoresis, LC-MS/MS, and stable isotope labeling by amino acids in cell culture (SILAC).

B cells play an essential role in the immune response. Upon activation they may differentiate into plasma cells that secrete specific antibodies against potentially pathogenic non-self antigens. To identify the cellular proteins that are important for efficient production of these antibodies we set out to study the B cell differentiation process at the proteome level. We performed an in-depth proteomic study to quantify dynamic relative protein expression patterns of several hundreds of proteins at five consecutive time points after lipopolysaccharide-induced activation of B lymphocytes. The proteome analysis was performed using a combination of stable isotope labeling using [13C6]leucine added to the murine B cell cultures, one-dimensional gel electrophoresis, and LC-MS/MS. In this study we identified 1,001 B cell proteins. We were able to quantify the expression levels of a quarter of all identified proteins (i.e. 234) at each of the five different time points. Nearly all proteins revealed changes in expression patterns. The quantitative dataset was further analyzed using an unbiased clustering method. Based on their expression profiles, we grouped the entire set of 234 quantified proteins into a limited number of 12 distinct clusters. Functionally related proteins showed a strong correlation in their temporal expression profiles. The quality of the quantitative data allowed us to even identify subclusters within functionally related classes of proteins such as in the endoplasmic reticulum proteins that are involved in antibody production.

Amino Acids↗
PubMed2004

Proteomics to diagnose human tumors and provide prognostic information.

Proteomics is a rapidly emerging scientific discipline that holds great promise in identifying novel diagnostic and prognostic biomarkers for human cancer. Technologic improvements have made it possible to profile and compare the protein composition within defined populations of cells. Laser capture microdissection is a tool for procuring pure populations of cells from human tissue sections to be used for downstream proteomic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used traditionally to separate complex mixtures of proteins. Improvements in this technology have greatly enhanced resolution and sensitivity providing a more reproducible and comprehensive survey. Image analysis software and robotic instrumentation have been developed to facilitate comparisons of complex protein expression patterns and isolation of differentially expressed proteins spots. Differential in-gel electrophoresis (DIGE) facilitates protein expression by labeling different populations of proteins with fluorescent dyes. Isotope-coded affinity tagging (ICAT) uses mass spectroscopy for protein separation and different isotope tags for distinguishing populations of proteins. Although in the past proteomics has been primarily used for discovery, significant efforts are being made to develop proteomic technologies into clinical tools. Reverse-phase protein arrays offer a robust new method of quantitatively assessing expression levels and the activation status of a panel of proteins. Surface-enhanced laser-desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy rapidly assesses complex protein mixtures in tissue or serum. Combined with artificial intelligence-based pattern recognition algorithms, this emerging technology can generate highly accurate diagnostic information. It is likely that mass spectroscopy-based serum proteomics will evolve into useful clinical tools for the detection and treatment of human cancers.

Early Diagnosis↗
PubMedDate not supplied

An isotope coding strategy for proteomics involving both amine and carboxyl group labeling.

This paper describes a heavy isotope coding strategy for the analysis of all types of tryptic peptides, including those that are N-terminally blocked and from the C-terminus of proteins. The method exploits differential derivatization of amine and carboxyl groups generated during proteolysis as a means of coding. Carboxyl groups produced during proteolysis incorporate 18O from H218O. Peptides from the C-terminus of proteins were not labeled with 18O unless they contained a basic C-terminal amino acid. Primary amines from control and experimental samples were differentially acylated after proteolysis with either 1H3- or 2H3-N-acetoxysuccinamide. When these two types of labeling were combined, unique coding patterns were achieved for peptides arising from the C-termini and blocked N-termini of proteins. This method was used to (1) distinguish C-terminal peptides in model proteins, (2) recognize N-terminal peptides from proteins in which the amino terminus is acylated, and (3) identify primary structure variations between proteins from different sources.

Amines↗
PubMed2006

Aberrantly regulated proteins in frontotemporal dementia.

Non-Alzheimer's disease of the frontal type, or frontotemporal dementia (FTD), is the second most common form of dementia. Yet, a detailed characterization of the disease has been especially limiting. To identify mechanisms possibly involved in disease pathology or progression, a proteomic analysis of proteins isolated from human frontal cortex with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) was performed. We used 2D gel electrophoresis and MALDI-TOF to identify a total of 24 proteins differentially expressed in FTDP-17. We identified a ubiquitin C-terminal hydrolase, UCHL1, as well as several proteins involved in oxidative stress to be differentially expressed. Data presented implicate UCHL1 and ubiquitin-mediated degradation as well as oxidative stress response in disease pathology or progression.

Chromosomes, Human, Pair 17↗
PubMed2006

Proteomic analysis of rat plasma by two-dimensional liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers therefore requires either the specific depletion of high abundance proteins using immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe a two-dimensional (2D) liquid chromatography separation method for the fractionation of rat plasma. In the first dimension proteins were separated by chromatofocusing according to their isoelectric point (pI). In the second dimension, proteins were further fractionated by non-porous, reversed-phase chromatography according to their hydrophobicity. The data from both separations was displayed as a 2D protein expression map of pI versus retention time (relative hydrophobicity). Both separations were carried out on the ProteomeLab PF 2D system (Beckman Coulter), an instrument platform that provides a high degree of automation and real-time monitoring of the separation process. The reproducibility of the first-dimension separation was evaluated in terms of pH gradient formation. The second-dimension separation was evaluated in terms of peak retention times on the reversed-phase column. We found in four consecutive chromatofocusing separations that the pH gradient differed by less than 0.2 pH units at any time during the elution step. Second dimension retention times of peaks from identical pI fractions differed by less than 7 s in six consecutive separations. Each 2D separation generated a total of 540 fractions which were analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). We detected approximately 275 peptides and proteins with molecular masses ranging from 3 to 225 kDa. Most fractions were found to contain multiple low and high molecular weight proteins. Differential display of 2D protein expression maps from retinol-sufficient and -deficient rat plasma samples identified a fraction with several proteins that appeared to be down-regulated in the vitamin A-deficient animal. Quantitative proteomic analysis of complex samples such as plasma is still a difficult task. We discuss the potential of this approach for biomarker discovery and address the experimental challenges that remain.

Animals↗
PubMed2026

Exploring the proteomic landscape of THP-1 monocytes through two-challenge LPS induction.

Proteome remodelling is central to the regulation of innate immune activation, yet the temporal organisation of protein networks engaged during repeated lipopolysaccharide (LPS) stimulation remains incompletely defined. In the present study, label-free quantitative mass spectrometry-based proteomics was used to characterise protein abundance changes in THP-1 monocytes at early (30 min) and later (2 h) time points following a second LPS challenge. This analysis was complemented by an independent co-immunoprecipitation proteomics experiment designed to identify candidate proteins associated with the regulatory pseudo-kinase IRAK3 during early TLR4 signalling. At 30 min, differentially abundant proteins were enriched in pathways associated with pattern-recognition receptor signalling, NF-κB activity, RNA processing, phosphorylation, and ribonucleoprotein complex organisation. By 2 h, the proteomic response broadened to include oxidative phosphorylation, antigen processing and presentation, vesicle-mediated transport, protein folding, and cytokine-regulatory pathways. These findings indicate that repeated LPS stimulation is accompanied by progressive remodelling of inflammatory, metabolic, translational, and proteostatic programmes rather than major changes in protein identity. Co-immunoprecipitation identified established TLR/IRAK3-associated components together with candidate IRAK3-associated proteins linked to RNA regulation, kinase signalling, ubiquitin-mediated processes, redox control, cytoskeletal remodelling, and damage-associated molecular pattern responses. Collectively, these findings define a temporal framework of proteomic adaptation during repeated inflammatory stimulation and expand the range of candidate proteins potentially contributing to IRAK3-centred regulation of innate immune signalling.

Humans↗
PubMed2024

Proteomic Heterogeneity of the Extracellular Matrix Identifies Histologic Subtype-Specific Fibroblast in Gastric Cancer.

Gastric cancer (GC) is a highly heterogeneous disease regarding histologic features, genotypes, and molecular phenotypes. Here, we investigate extracellular matrix (ECM)-centric analysis, examining its association with histologic subtypes and patient prognosis in human GC. We performed quantitative proteomic analysis of decellularized GC tissues that characterizes tumorous ECM, highlighting proteomic heterogeneity in ECM components. We identified 20 tumor-enriched proteins including four glycoproteins, serpin family H member 1 (SERPINH1), annexin family (ANXA3/4/5/13), S100A family (S100A6/8/9), MMP14, and other matrisome-associated proteins. In addition, histopathological characteristics of GC reveals differential expression in ECM composition, with the poorly cohesive carcinoma-not otherwise specified (PCC-NOS) subtype being distinctly demarcated from other histologic subtypes. Integrating ECM proteomics with single-cell RNA sequencing, we identified crucial molecular markers in the PCC-NOS-specific stroma. PCC-NOS-enriched matrisome proteins and gene expression signatures of adipogenic cancer-associated fibroblasts (CAFadi) are closely linked, both associated with adverse outcomes in GC. Using tumor microarray analysis, we confirmed the CAFadi surface marker, ATP binding cassette subfamily A member 8 (ABCA8), predominantly present in PCC-NOS tumors. Our ECM-focused analysis paves the way for studies to determine their utility as biomarkers for patient stratification, offering valuable insights for linking molecular and histologic features in GC.

Humans↗
PubMedDate not supplied

Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and cICAT with multidimensional liquid chromatography and tandem mass spectrometry.

A total of nine potential markers for endometrial cancer (EmCa) have been discovered and identified from endometrial tissue homogenates using a combination of differentially labeled tags, iTRAQ and cICAT, with multidimensional liquid chromatography and tandem mass spectrometry. The tissues were snap frozen in liquid nitrogen within 15-20 min after devitalization. Samples for proteomic analysis were treated with protease inhibitors before processing. Marker proteins that were overexpressed in EmCa are chaperonin 10, pyruvate kinase M1 or M2 isozyme, calgizzarin, heterogeneous nuclear ribonucleoprotein D0, macrophage migratory inhibitory factor, and polymeric immunoglobulin receptor precursor; those that were underexpressed are alpha-1-antitrypsin precursor, creatine kinase B, and transgelin. The chaperonin 10 result confirms our earlier observation of overexpression in EmCa tissues using surface-enhanced laser desorption/ionization mass spectrometry, verified by Western analysis and immunohistochemistry [Yang, E. C. C. et al. J. Proteome Res. 2004, 3, 636-643]. Pyruvate kinase was observed to be overexpressed using both iTRAQ and cICAT labeling. All nine markers have been found to be associated with various forms of cancer. A panel of these plus other markers may confer sufficient selectivity for diagnosing and screening of EmCa. The use of cICAT led to identification of a higher proportion of lower-abundance signaling proteins; conversely, iTRAQ resulted in a higher percentage of the more abundant ribosomal proteins and transcription factors.

Chromatography, Liquid↗
PubMed2006

Inhibitory effects of procyanidin B(2) dimer on lipid-laden macrophage formation.

A proteomic analysis of procyanidin B(2) isolated from cocoa against oxidized low-density lipoprotein-induced lipid-laden macrophage formation was performed. Of approximately 400 detected proteins, 12 were differentially expressed as a result of B(2) treatment. They were subsequently identified by liquid chromatography-electrospray ionization-tandem mass spectrometry and the SWISS-PROT database. Further reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that B(2) strongly inhibited arachidonic acid inflammatory reactions, apoptosis, and their coupled mitogen-activated protein kinase and NF-kappaB pathways. To highlight proteins or genes with similar expressed patterns and similarly biological function induced by B(2) in lipid-laden macrophages, a cluster and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed. The data were mapped to multiple pathways. Further validation of the bioinformatic results revealed that activation of Wnt signaling may contribute to the cardioprotection of B(2). The differentially expressed genes and proteins mentioned above induced by B(2) are through regulating nuclear transcription factors, activating peroxisome proliferator-activated receptor-gamma and inhibiting AP-1 mRNA expressions. These in vitro data help to interpret the beneficial effects of B(2) in reducing the risk of atherosclerosis after consumption of flavonoid-rich foods. Many differentially expressed genes induced by B(2) help to uncover novel targets and may help to target disease interactions in atherosclerosis in the future.

Amino Acid Sequence↗
PubMed2006

Identification of the proliferation/differentiation switch in the cellular network of multicellular organisms.

The protein-protein interaction networks, or interactome networks, have been shown to have dynamic modular structures, yet the functional connections between and among the modules are less well understood. Here, using a new pipeline to integrate the interactome and the transcriptome, we identified a pair of transcriptionally anticorrelated modules, each consisting of hundreds of genes in multicellular interactome networks across different individuals and populations. The two modules are associated with cellular proliferation and differentiation, respectively. The proliferation module is conserved among eukaryotic organisms, whereas the differentiation module is specific to multicellular organisms. Upon differentiation of various tissues and cell lines from different organisms, the expression of the proliferation module is more uniformly suppressed, while the differentiation module is upregulated in a tissue- and species-specific manner. Our results indicate that even at the tissue and organism levels, proliferation and differentiation modules may correspond to two alternative states of the molecular network and may reflect a universal symbiotic relationship in a multicellular organism. Our analyses further predict that the proteins mediating the interactions between these modules may serve as modulators at the proliferation/differentiation switch.

Animals↗
PubMed2006

New multi protein patterns differentiate liver fibrosis stages and hepatocellular carcinoma in chronic hepatitis C serum samples.

AIM: To identify a multi serum protein pattern as well as single protein markers using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) for detection and differentiation of liver fibrosis (F1-F2), liver cirrhosis (F4) and hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus (HCV). METHODS: Serum samples of 39 patients with F1/F2 fibrosis, 44 patients with F4 fibrosis, 34 patients with HCC were applied to CM10 arrays and analyzed using the SELDI-TOF ProteinChip System (PBS-IIc; Ciphergen Biosystems) after anion-exchange fractionation. All patients had chronic hepatitis C and histologically confirmed fibrosis stage/HCC. Data were analyzed for protein patterns by multivariate statistical techniques and artificial neural networks. RESULTS: A 4 peptide/protein multimarker panel (7486, 12,843, 44,293 and 53,598 Da) correctly identified HCCs with a sensitivity of 100% and specificity of 85% in a two way-comparison of HCV-cirrhosis versus HCV-HCC training samples (AUROC 0.943). Sensitivity and specificity for identification of HCC were 68% and 80% for random test samples. Cirrhotic patients could be discriminated against patients with F1 or F2 fibrosis using a 5 peptide/protein multimarker pattern (2873, 6646, 7775, 10,525 and 67,867 Da) with a specificity of 100% and a sensitivity of 85% in training samples (AUROC 0.976) and a sensitivity and specificity of 80% and 67% for random test samples. Combination of the biomarker classifiers with APRI score and alfa-fetopotein (AFP) improved the diagnostic performance. The 6646 Da marker protein for liver fibrosis was identified as apolipoprotein C-I. CONCLUSION: SELDI-TOF-MS technology combined with protein pattern analysis seems a valuable approach for the identification of liver cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis C. Most probably a combination of different serum markers will help to identify liver cirrhosis and early-stage hepatocellular carcinomas in the future.

Adult↗
PubMed2006

Proteomic analysis of p38alpha mitogen-activated protein kinase-regulated changes in membrane fractions of RAS-transformed fibroblasts.

Oncogenic Ras signaling has been long known to play an important role in tumorigenesis and human cancer. In this report, we have used the sensitive 2-D-DIGE coupled to MS for the identification of proteins differentially expressed at the cell membrane level between oncogenic H-RasV12-transformed wild-type and p38alpha-deficient mouse embryo fibroblasts (MEFs). Following trifluoroethanol solubilization, 76 proteins were found to be differentially regulated. After PMF, 63 spots containing 42 different proteins were unequivocally identified by MALDI-TOF MS coupled with database interrogation. As expected, many of them were membrane proteins. Six proteins were selected for further validation studies based on their potential functional link with malignant transformation and signal transduction. These were prohibitin (PHB), protein disulfide isomerase 3 (PDIA3), focal adhesion kinase 2 (FAK2), c-GMP dependent protein kinase 2 (KGP2), NADH-ubiquinone oxidoreductase 30 kDa subunit (NUGM) and translationally controlled tumor protein (TCTP). All these proteins were up-regulated in the membranes of H-RasV12-transformed p38alpha-/-cells, except for prohibitin, which was down-regulated. An excellent correlation was found between DIGE results and Western blot studies, indicating the reliability of the 2-D-DIGE analysis. The available evidence about the putative function of the identified proteins supports the emerging role of p38alpha as a negative regulator of tumorigenesis. Further studies are in progress to elucidate the implications of these findings in the regulation of H-Ras-induced transformation by p38alpha signaling.

Animals↗
PubMed2006

Comparative proteomic analysis to study molecular events during gonad development in mice.

Sex determination represents a critical bifurcation in the road of embryonic development. It is based on a finely regulated network of gene activity, as well as protein-protein interactions and activation or silencing of signaling pathways. Despite the identification of a number of critical genes, many aspects of the molecular cascade that drives the differentiation of the embryonic gonad into either a testis or an ovary remain poorly understood. To identify new proteins involved in this cascade, we employed two-dimensional gel electrophoresis and mass spectrometry to compare the protein expression profiles of fetal mouse testes and ovaries. Three proteins, hnRPA1, TRA1, and HSC71, were found to be expressed in a male-specific manner and this expression was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Moreover, HSC71 was found to be hyperphosphorylated in male compared to female gonads, emphasizing the advantage of the proteomic approach in allowing the detection of posttranslational modifications.

Animals↗
PubMed2002

Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways.

Injury to motor neurons associated with mutant Cu,Zn-superoxide dismutase (SOD1)-related familial amyotrophic lateral sclerosis (FALS) results from a toxic gain-of-function of the enzyme. The mechanisms by which alterations to SOD1 elicit neuronal death remain uncertain despite intensive research effort. Analysis of the cellular proteins that are differentially expressed in the presence of mutant SOD1 represents a novel approach to investigate further this toxic gain-of-function. By using the motor neuron-like cell line NSC34 stably transfected with wild-type, G93A, or G37R mutant human SOD1, we investigated the effects of mutant human SOD1 on protein expression using proteomic approaches. Seven up-regulated proteins were identified as argininosuccinate synthase, argininosuccinate lyase, neuronal nitric-oxide synthase, RNA-binding motif protein 3, peroxiredoxin I, proteasome subunit beta 5 (X), and glutathione S-transferase (GST) Alpha 2. Seven down-regulated proteins were identified as GST Mu 1, GST Mu 2, GST Mu 5, a hypothetical GST Mu, GST Pi B, leukotriene B(4) 12-hydroxydehydrogenase, and proteasome subunit beta5i (LMP7). GST assays demonstrated a significant reduction in the total GST activity of cells expressing mutant human SOD1. Proteasome assays demonstrated significant reductions in chymotrypsin-like, trypsin-like, and post-glutamylhydrolase proteasome activities. Laser capture microdissection of spinal cord motor neurons from human FALS cases, in conjunction with reverse transcriptase-PCR, demonstrated decreased levels of mRNA encoding GST Mu 1, leukotriene B(4) 12-hydroxydehydrogenase, and LMP7. These combined approaches provide further evidence for involvement of alterations in antioxidant defenses, proteasome function, and nitric oxide metabolism in the pathophysiology of FALS.

Amino Acid Substitution↗
PubMed2002

IL-1beta induced protein changes in diabetes prone BB rat islets of Langerhans identified by proteome analysis.

AIMS/HYPOTHESIS: Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1beta (IL-1beta) modulates the beta-cell function, protein synthesis, energy production and causes apoptosis. We have previously shown changes in the expression of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1beta exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen in IL-1beta exposed Wistar Furth (WF) rat islets. METHODS: The 82 protein spots, that changed expression after IL-1beta exposure, were all re-identified on preparative gels of 200 000 neonatal WF rat islets, cut out and subjected to mass spectrometry for identification. RESULTS: Forty-five different proteins were identified from 51 spots and grouped according to function: (i) energy transduction and redox potentials; (ii) glycolytic and Krebs cycle enzymes; (iii) protein, DNA and RNA synthesis, chaperoning and protein folding; (iv) signal transduction, regulation, differentiation and apoptosis; (v) cellular defence; and (vi) other functions. Comparison of IL-1beta exposed BB-DP and WF islets showed common changes in 14 proteins and several proteins influencing similar pathways, suggesting that similar routes in the two strains lead to beta-cell destruction. CONCLUSION/INTERPRETATION: We demonstrate that proteome analysis is a powerful tool to identify proteins and pathways in BB-DP rat islets exposed to IL-1beta.

Animals↗
PubMed2003

Proteomic analysis of oxidative stress-resistant cells: a specific role for aldose reductase overexpression in cytoprotection.

We are using a proteomic approach that combines two-dimensional electrophoresis and tandem mass spectrometry to detect and identify proteins that are differentially expressed in a cell line that is resistant to oxidative stress. The resistant cell line (OC14 cells) was developed previously through chronic exposure of a parent cell line (HA1 cells) to increasing hydrogen peroxide concentrations. Biochemical analyses of this system by other investigators have identified elevated content and activity of several classical antioxidant proteins that have established roles in oxidative stress resistance, but do not provide a complete explanation of this resistance. The proteomics studies described here have identified the enzyme aldose reductase (AR) as 4-fold more abundant in the resistant OC14 cells than in the HA1 controls. Based on this observation, the role of AR in the resistant phenotype was investigated by using a combination of AR induction with ethoxyquin and AR inhibition with Alrestatin to test the cytotoxicity of two oxidation-derived aldehydes: acrolein and glycolaldehyde. The results show that AR induction in HA1 cells provides protection against both acrolein- and glycolaldehyde-induced cytotoxicity. Furthermore, glutathione depletion sensitizes the cells to the acrolein-induced toxicity, but not the glycolaldehyde-induced toxicity, while AR inhibition sensitizes the cells to both acrolein- and glycolaldehyde-induced. These observations are consistent with a significant role for AR in the oxidative stress-resistant phenotype. These studies also illustrate the productive use of proteomic methods to investigate the molecular mechanisms of oxidative stress.

Acrolein↗
PubMed2006

Proteomic approach to substrates related to MAPK pathway in 293T cells.

Abundant evidence indicates that potential scaffold proteins and adaptor or linker molecules organize and specify various MAP kinase cascades. In the present study, proteomic methodologies were applied to screen these potential molecules in combination with cell morphology and cell cycle analysis. MEK1E, MKK3b, MKK5D and MKK7D were selected as representative MKKs of four main MAPK pathways. Our results showed that similar morphological transformation and G(2)/M cell cycle arrest were promoted by the over-expressed four kinases. Furthermore, global change in response to the over-expressed four kinases was characterized by differential proteomics. Eleven distinctly changed proteins were detected, in which four proteins (serine/threonine kinase 4, glutathione S-transferase p1-1, glycoprotein IX and soluble inorganic pyrophosphatase) were reported to be relative to MAPK pathways, while the other seven proteins may be new elements of substrates of the kinases. In our experiment, the expression of platelet glycoprotein IX precursor, glutathione S-transferase p1-1, peroxiredoxin 6, Ras-related protein Rab-34 and arginase II, mitochondrial precursor was up-regulated, while the expression of serine/threonine kinase 4 (MST1) was down-regulated by the four kinases. These results suggest that these six proteins may be common targets of all the MAPK pathways in 293T cell line. Interestingly, the expression of splicing factor 3B subunit 4 and soluble inorganic pyrophosphatase (Ppase) was specifically up-regulated by MEK1E and MKK5D, and by MEK1E, MKK3b and MKK5D, respectively. The expression of methylglyoxalase was down-regulated by MEK1E and MKK7D. Furthermore, the expression of ADP-ribosylation factor-like protein 1 was up-regulated by MKK5D but down-regulated by MKK3b and MKK7D. These findings revealed the characteristic molecular responses to four MKKs. In conclusion, our study not only confirms that MST1, glutathione S-transferase p1-1, glycoprotein IX and soluble PPase belong to MAPK pathways, but also provides seven novel molecules for the further study of the pathways.

Cell Cycle↗
PubMed2004

Protein patterns of pig oocytes during in vitro maturation.

In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. In this study, we have launched the analysis of pig oocytes during in vitro maturation using a proteomics approach. First, oocyte proteins have been separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Remarkably, several proteins, including peroxiredoxins, ubiquitin carboxyl-terminal hydrolase isozyme L1, and spermine synthase, are even more abundant than actin, usually the most abundant protein in somatic cells. Furthermore, we have initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins, which are differentially expressed during in vitro maturation. To date, we have identified antiquitin (D7A1), the member of aldehyde dehydrogenase family7 that has been significantly increased in MI and MII stages compared with GV oocytes. To our knowledge, this is the first pig oocyte proteome available so far that may be used as a reference map. The proteins that are differentially regulated during IVM may present potential biomarkers of oocyte maturation and quality. It is a useful inventory toward a deeper understanding of the mechanisms underlying reproduction and development.

Animals↗