Comparative proteomic analysis associated with term placental insufficiency in cloned pig

Somatic cell-derived nuclear transfer (scNT) is a method of animal cloning in which the oocyte reprograms a somatic cell nucleus to divide and execute developmental programs. Despite many successes in this field, cloning by scNT remains very inefficient. Unlike other cloned animals, pigs derived by scNT have placentas with severe villous hypoplasia. To obtain a better understanding of the protein networks involved in this phenomenon, we assessed global protein expression profiles in term placentas from scNT-derived and control animals. Proteomic analysis of term placentas from scNT-derived animals identified 43 proteins that were differentially expressed compared to control animals. Among them, 14-3-3 proteins and Annexin V, which are closely involved in the apoptotic signaling pathway, were significantly down- and up-regulated, respectively. Western blot analysis and immunohistochemistry indicated that down-regulation of 14-3-3 proteins in scNT-derived placentas induced apoptosis of cytotrophoblast cells via mitochondria-mediated apoptosis. Taken together, our results suggest that placental insufficiency in scNT-derived placentas may be due to apoptosis, induced in part by the down-regulation of 14-3-3 proteins and up-regulation of Annexin V. They also indicate that proteomic maps represent an important tool for future studies of placental insufficiency and pathology.     

Isolation of genes differentially expressed during the fruit body development of Pleurotus ostreatus by differential display of RAPD

To analyze genes involved in fruit body development of Pleurotus ostreatus, mRNAs from three different developmental stages: i.e., vegetative mycelium, primordium, and mature fruit body, were isolated and reverse-transcribed to cDNAs. One hundred and twenty random PCR amplifications were performed with the cDNAs, which generated 382, 394, 393 cDNA fragments from each developmental stage. From these fragments, four cDNA clones specifically expressed in primordium or mature fruit body were detected. Sequence analysis and database searches revealed significant similarity with triacylglycerol lipase, cytochrome P450 sterol 14 alpha-demethylase and developmentally regulated genes of other fungi. Northern blot analyses confirmed that all of the four cDNAs were unexpressed in mycelium, thus stage-specific genes for fruit body formation of P. ostreatus were successfully isolated.     

Differential proteomic analysis of proteins in wheat spikes induced by Fusarium graminearum

Scab, caused by Fusarium graminearum, is a serious spike disease in wheat. To identify proteins in resistant wheat cultivar Wangshuibai induced by F. graminearum infection, proteins extracted from spikes 6, 12 and 24 h after inoculation were separated by 2-DE. Thirty protein spots showing 3-fold change in abundance when compared with treatment without inoculation were characterized by MALDI-TOF MS and matched to proteins by querying the mass spectra in protein databases or the Triticeae EST translation database. Based on their volume profiles, these proteins were classified into four categories. The first one fell off rapidly at the initial inoculation and then rose at 12 or 24 hai, the second one decreased considerably after inoculation and remained at low level, the third one rose at the initial inoculation and then declined at 12 or 24 hai, the forth one showed steady increase after inoculation and maintained at a high level. Many of the proteins identified in the first two categories are related to carbon metabolism and photosynthesis. While most of proteins identified in the last two categories are related to stress defense of plants, indicating that proteins associated with the defense reactions were activated or translated shortly after inoculation.     

Identification of differentially expressed proteins in Ficedula flycatchers

This is the first report on the large-scale identification and comparison of proteins in non-model organisms, Ficedula flycatchers. It highlights the potential of proteomics approaches in both non-sequenced and non-model organisms for identification of differentially expressed proteins. Not surprisingly, more than 55% of the proteins failed to be identified even though the MS spectra were of high quality. Nevertheless, the protein information obtained in this study will serve as a valuable resource for continued research.     

Comparative proteomic analysis of human placenta derived from assisted reproductive technology

The aim of this study was to use proteomics-based approach to examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Using 2-DE we found that, compared with the control group, 12 spots in standard in vitro fertilization group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differentially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, alpha-SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot and immunohistochemistry analysis. Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.     

Investigation of differentially expressed genes during the development of mouse cerebellum

Before the discovery of DNA microarray and DNA chip technology, the expression of only a small number of genes could be analyzed at a time. Currently, such technology allows us the simultaneous analysis of a large number of genes to systematically monitor their expression patterns that may be associated with various biological phenomena. We utilized the Affymetrix GeneChip Mu11K to analyze the gene expression profile in developing mouse cerebellum to assist in the understanding of the genetic basis of cerebellar development in mice. Our analysis showed 81.6% (10,321/12,654) of the genes represented on the GeneChip were expressed in the postnatal cerebellum, and among those, 8.7% (897/10,321) were differentially expressed with more than a two-fold change in their maximum and minimum expression levels during the developmental time course. Further analysis of the differentially expressed genes that were clustered in terms of their expression patterns and the function of their encoded products revealed an aspect of the genetic foundation that lies beneath the cellular events and neural network formation that takes place during the development of the mouse cerebellum.     

Alternative and effective proteomic analysis in Arabidopsis

Various functional genomics platforms are required to define the phenotype associated with a mutant. Global protein analyses may be included in any study. We describe here a rapid method of protein sample preparation and analysis, suitable for all laboratories and using Arabidopsis plantlets as the starting material. This reliable and reproducible method for high yield protein extraction from small amounts of material can be used on even the most recalcitrant tissues. The proteins extracted are suitable for many types of protein analysis, including nondenaturing investigations. This method was validated by a rigorous 2-DE approach, coupled with unambiguous LC-MS/MS identifications featuring strong sequence coverage (average of 26% with eight different peptides/spot protein). The reproducibility of the method was demonstrated by multiple protein identifications from identical series of spots. An interactive map (http://www.isv.cnrsgif.fr/gel2d/), including 435 protein variants showed that (i) 38% of the proteins were yet unreported, (ii) reduced subfractionation, (iii) had frequent protein modifications (average of two spots/protein entry), and (iv) underwent no major proteolytic events other than leader peptide cleavage. Finally, a simple mobility shift method for the large subunit of RuBisCo (LS) in the first dimension made it possible to characterize previously masked protein spots.     

Pigment epithelium-derived factor is differentially expressed in peripheral neuropathies

Peripheral neuropathies are characterized by asymmetrical slowly progressive weakness with no upper motor neuron signs, and can occur either with or without pain. Due to poor knowledge of the disease mechanisms, available pain treatment is very limited. Because of the difficulties and invasiveness involved when performing direct analysis on peripheral and CNS, pathological markers can be searched for in the cerebrospinal fluid (CSF) as an alternative. To investigate pain mechanisms in peripheral neuropathy and find diagnostic markers, CSF samples were analyzed by a differential expression proteomic approach. We studied CSF from: neuropathic patients with pain (PN), without pain (NPN) and healthy controls (CN). 2-DE analysis showed ten protein spots differentially expressed, and six of these were identified by MS. In NPN patients we found an expression level decrease of three pigment epithelium-derived factor (PEDF) protein isoforms. Immunoblot with a specific antibody revealed the presence of additional PEDF isoforms not highlighted by differential expression analysis. Fucose residues on the oligosaccharide chain were found only in the isoforms down regulated in NPN patients. Considered as PEDF has important neurobiological effects, it might be considered an interesting pathology marker.     

Search for the tumor-related proteins of transition cell carcinoma in Taiwan by proteomic analysis

To better understand the carcinogenesis of bladder cancer in Taiwan, we utilized the proteomic approach to search for potential biomarkers of transitional cell carcinoma (TCC). Analysis by 2-DE and MS/MS indicated that seven proteins are down-regulated and three proteins up-regulated in grade III samples as compared with those of grade II. Of these deregulated proteins, fatty acid binding proteins, annexin V, heat-shock protein 27, and lactate dehydrogenase have been shown to be associated with bladder cancer. Our studies also found altered expression of a group of proteins that have not been documented previously in bladder cancer, including annexin I, 15-hydroxyprostaglandin dehydrogenase, galectin-1, lysophospholipase and mitochondrial short-chain enoyl-coenzyme A hydratase 1 precursor. These results illustrate a pattern of differential protein expression between low- and high-grade tumors and it may be utilized as the molecular fingerprinting of a subset of bladder cancers. In addition, the present study provides a valuable resource in the study of pathological mechanisms in cancers of urothelial origin. The immunohistochemical staining of grade II and III TCC samples with antiserum to annexin I protein was utilized to confirm that the annexin I protein is up-regulated in grade III TCC.     

Protein extraction from plant tissues for 2DE and its application in proteomic analysis

Plant tissues contain large amounts of secondary compounds that significantly interfere with protein extraction and 2DE analysis. Thus, sample preparation is a crucial step prior to 2DE in plant proteomics. This tutorial highlights the guidelines that need to be followed to perform an adequate total protein extraction before 2DE in plant proteomics. We briefly describe the history, development, and feature of major sample preparation methods for the 2DE analysis of plant tissues, that is, trichloroacetic acid/acetone precipitation and phenol extraction. We introduce the interfering compounds in plant tissues and the general guidelines for tissue disruption, protein precipitation and resolubilization. We describe in details the advantages, limitations, and application of the trichloroacetic acid/acetone precipitation and phenol extraction methods to enable the readers to select the appropriate method for a specific species, tissue, or cell type. The current applications of the sample preparation methods in plant proteomics in the literature are analyzed. A comparative proteomic analysis between male and female plants of Pistacia chinensis is used as an example to represent the sample preparation methodology in 2DE‐based proteomics. Finally, the current limitations and future development of these sample preparation methods are discussed. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP17).     

A proteomic approach to identify differentially expressed plasma proteins between the fed and prolonged fasted states

Prolonged fasting is characterized by consecutive phases, a short period of adaptation (phase 1), phase 2 (P2) characterized by fat oxidation, and phase 3 (P3) during which energy requirements are mostly derived from increased protein utilization. At this latter stage, food seeking behavior is induced. Very few circulating biomolecules have been identified that are involved in the response to prolonged fasting. To this end, rat plasma samples were compared by a proteomic approach, using 2‐DE. The results revealed a selective variation of the levels of apolipoprotein A‐IV, A‐I, and E, haptoglobin, transthyretin, plasma retinol binding‐protein, and vitamin D binding‐protein in P2 and P3. The variations in protein levels were confirmed by ELISA. Changes in mRNA levels encoding these proteins did not systematically correlate well with protein concentrations, and tissue‐specific regulation of mRNA expression was observed, underlining the complex metabolic regulation in response to food deprivation. In late fasting, the marked reduction of apolipoprotein A‐IV levels could contribute to the alarm signal that triggers refeeding. The variations of the other differentially expressed proteins are more likely related to lipid metabolism and insulin signaling alterations.     

Zinc adaptation and resistance to cadmium toxicity in mammalian cells: molecular insight by proteomic analysis

To identify proteins involved in cellular adaptive responses to zinc, a comparative proteome analysis between a previously developed high zinc- and cadmium-resistant human epithelial cell line (high zinc-resistant HeLa cells, HZR) and the parental HeLa cells has been carried out. Differentially produced proteins included cochaperones, proteins associated with oxido-reductase activities, and ubiquitin. Biochemical pathways to which these proteins belong were probed for their involvement in the resistance of both cell lines against cadmium toxicity. Among ER stressors, thapsigargin sensitized HZR cells, but not HeLa cells, to cadmium toxicity more acutely than tunicamycin, implying that these cells heavily relied on proper intracellular calcium distribution. The similar sensitivity of both HeLa and HZR cells to inhibitors of the proteasome, such as MG-132 or lactacystin, excluded improved proteasome activity as a mechanism associated with zinc adaptation of HZR cells. The enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) was overproduced in HZR cells as compared to HeLa cells. It transforms HPP to homogentisate in the second step of tyrosine catabolism. Inhibition of HPPD decreased the resistance of HZR cells against cadmium, but not that of HeLa cells, suggesting that adaptation to zinc overload and increased HPP removal are linked in HZR cells.     

Presenilin 1 and 2 are expressed differentially in the cerebral cortex of mice during development

Presenilin (PS) 1 and PS2 are multi-pass transmembrane proteins involved in vital brain functions. Studies using transgenic or conditional knockout models show that PS1 is implicated in crucial brain developmental processes. Conversely, PS2 knockout mice do not exhibit any abnormality in the brain morphology, suggesting that PS2 may not be involved in brain development. However, there is no holistic information available for endogenous expression of PS during brain development. Therefore, we have examined the distribution and expression profile of PS1 and PS2 mRNA and protein in the cerebral cortex of prenatal, neonatal and postnatal mice. The results revealed that the distribution and expression profile of PS1 and PS2 mRNA varied significantly in the cerebral cortex during development. In prenatal stages, both PS1 and PS2 mRNA showed high expression at embryonic day (E) 12.5 and downregulation at E18.5. Postnatally, PS1 mRNA showed upregulation from postnatal day 0 (P0) to P45 and thereafter reduction at 20weeks, but PS2 mRNA showed no significant alteration. However, they did not exhibit any significant regional variation except at E18.5, when PS2 showed reduction in temporal and medial temporal lobes as compared to frontal and parietal lobes. Furthermore, PS1 showed significant change in protein expression similar to its mRNA profile. However, PS2 protein expression did not correspond to its mRNA; it was highest at E12.5, downregulated up to P20 and then upregulated at P45 and 20weeks. Taken together, our study demonstrates for the first time that the distribution and expression profile of PS2 is different from PS1 in the mouse cerebral cortex during development.