The human pituitary nitroproteome: detection of nitrotyrosyl-proteins with two-dimensional Western blotting, and amino acid sequence determination with mass spectrometry

Nitric oxide is an important mediator that participates in reduction-oxidation (redox) mechanisms and in cellular signal transduction pathways. Two types of post-translational modifications are induced by nitric oxide: S-nitrosylation of cysteine residues and nitration of tyrosine residues. Two-dimensional gel electrophoresis-based Western blotting was used to detect, and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) to determine the amino acid sequence of, several different nitrated proteins in the human pituitary. Proteins from several 2D gel spots, which corresponded to the strongly positive anti-nitrotyrosine Western blot spots, were subjected to in-gel trypsin-digestion and LC-MS/MS analysis. MS/MS, SEQUEST analysis, and de novo sequencing were used to determine the nitration site of each nitrated peptide. A total of four different nitrated peptides were characterized and were matched to four different proteins: synaptosomal-associated protein, actin, immunoglobulin alpha Fc receptor, and cGMP-dependent protein kinase 2. Those nitrotyrosyl-proteins participate in neurotransmission, cellular immunity, and cellular structure and mobility.     

The holm oak leaf proteome: analytical and biological variability in the protein expression level assessed by 2-DE and protein identification tandem mass spectrometry de novo sequencing and sequence similarity searching

As a first approach in establishing the holm oak leaf proteome, we have optimised a protocol for this plant and tissue which includes the following steps: trichloroacetic acid-acetone extraction, two-dimensional gel electrophoresis (2-DE) on pH 5 to 8 linear gradient immobilised pH gradient strips as the first dimension, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% polyacrylamide gels as the second one. Proteins were detected by Coomassie staining. Gel images were recorded and digitalized, and the protein spots quantified by using a linear regression equation of protein quantity on spot volume obtained against standard proteins. Analytical variance was calculated for one-hundred protein spots from three replicate 2-DE gels of the same protein extract. Biological variance was determined for the same protein spots from independent tissue extracts corresponding to leaves from different trees, or the same tree at different orientations or sampling times during a day. Values of 26% for the analytical variance and 58.6% for the biological variance among independent trees were obtained. These values provide a quantified and statistical basis for the evaluation of protein expression changes in comparative proteomic investigations with this species. A representative set of the major proteins, covering the isoelectric point range of 5 to 8 and the relative molecular mass(r) range of 14 to 78 kDa, were subjected to liquid chromatography-tandem mass spectrometry analysis. Due to the absence of Quercus DNA or protein sequence databases, a method based on the procedure reported by Liska and Shevchenko including de novo sequencing and BLAST similarity searching against other plant species databases was used for protein identification. Out of 43 analysed spots, 35 were positively identified. The identified proteins mainly corresponded to enzymes involved in photosynthesis and energetic metabolism, with a significant number corresponding to RubisCO.     

Identification of 4-hydroxynonenal-modified retinal proteins induced by photooxidative stress prior to retinal degeneration

4-Hydroxynonenal (4-HNE) is a reactive aldehyde species generated endogenously from the nonenzymatic oxidation of n-6 polyunsaturated fatty acids under physiological conditions. We have reported that intense white light exposure increases 4-HNE-protein modification in the retina prior to the onset of photoreceptor cell apoptosis. To understand the molecular mechanism(s) underlying the retinal degeneration induced by photooxidative stress, we identified 4-HNE-modified retinal proteins using a proteomic approach. Albino rats were exposed to 5 k lx white fluorescent light for 3 h and retinas were removed 24 h later and pooled. By Western dot blot analysis, the total intensity of 4-HNE-modified proteins was increased 1.5-fold following the exposure compared to dim light controls. In two independent sets of two-dimensional gel electrophoresis/Western blots followed by peptide mass fingerprinting (PMF), nine proteins including voltage-dependent anion channel, enolase 1, aldolase C, crystallins A and βB3, heterogeneous nuclear ribonucleoprotein A2/B1, albumin, and glutamine synthetase were identified. We observed that 4-HNE modifications of retinal proteins are specific to a particular set of proteins rather than random events on abundant proteins. By immunohistochemistry, localization of 3 identified proteins overlapped with immunoreactivity of 4-HNE-modified proteins in light-exposed retinas. Intense light exposure increases 4-HNE-protein modifications on specific retinal proteins in several functional categories including energy metabolism, glycolysis, chaperone, phototransduction, and RNA processing. Together with previous reports that 4-HNE modification changes protein activities, these results suggest a close association of 4-HNE-protein modifications with the initiation of light-induced retinal degeneration.     

Salivary protein changes in response to acute stress in medical residents performing advanced clinical simulations: a pilot proteomics study

Context: Quantitative changes of salivary proteins due to acute stress were detected.

Objective: To explore protein markers of stress in saliva of eight medical residents who performed emergency medicine simulations.

Materials and methods: Saliva was collected before the simulations, after the simulations, and following morning upon waking. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), identified by mass spectrometry (MS), and relatively quantified by densitometry.

Results: Salivary alpha-amylase and S–type cystatins significantly increased, while the ～26?kDa and low-molecular weight (MW) (<10?kDa) SDS-PAGE bands exhibited changes after stress.

Discussion and conclusion: Alpha-amylase and cystatins are potential salivary markers of acute stress, but further validation should be performed using larger sample populations.     

Down-regulation of the strawberry Bet v 1-homologous allergen in concert with the flavonoid biosynthesis pathway in colorless strawberry mutant

Proteomic screening of strawberry (Fragaria ananassa) yielded a 58% success rate in protein identification in spite of the fact that no genomic sequence is available for this species. This was achieved by a combination of MALDI-MS/MS de novo sequencing of double-derivatized peptides and indel-tolerant searching against local protein databases built on both EST and full-length nucleotide sequences. The amino acid sequence of a strawberry allergen, homologous to the well-known major birch pollen allergen Bet v 1, was partially determined. This strawberry allergen, named Fra a 1 according to the nomenclature for allergen proteins, showed sequence identity of 54 and 77%, respectively, with corresponding allergens from birch and apple. Differential expression, as evaluated by 2-D DIGE, occurred in 10% of protein spots when red strawberries were compared to a colorless (white) strawberry mutant. White strawberries, known to be tolerated by individuals affected by allergy, were found to be virtually free from the strawberry allergen. Also several enzymes in the pathway for biosynthesis of flavonoids, to which the red color pelargonidin belongs, were down-regulated. This approach to assess differential protein expression without access to genomic sequence information can also be applied to other crop plants and phenotypic traits.     

Transient repression of the synthesis of OmpF and aspartate transcarbamoylase in Escherichia coli K12 as a response to pollutant stress

Abstract The synthesis of total cellular proteins in Escherichia coli K12 was studied in batch culture following exposure of cells to low concentrations of monochlorophenol, pentachlorophenol and cadmium chloride. Changes in protein patterns were identified after pulse-chase labelling of proteins with [³⁵S]methionine and subsequent two-dimensional gel electrophoresis (2D-PAGE). We demonstrated that besides the induction of some stress proteins, also a transient decrease in the rate of synthesis of other proteins occurred. Two of these proteins were identified as OmpF and aspartate transcarbamoylase (ATCase). Their transient repression appeared to be a general response to stress elicited by different pollutants and may therefore be used as a general and sensitive early warning system for pollutant stress.     

Domestication is associated with differential expression of pikeperch egg proteins involved in metabolism,immune response and protein folding

Domestication is a condition in which the breeding, care and feeding of animals are, at least in part, controlled by humans. Information regarding the changes in the protein composition of eggs in response to domestication is very limited. Such data are prerequisite for improvements in the reproduction of domesticated fish. The aim of this study was to examine the impact of domestication on the proteome of pikeperch eggs using two-dimensional differential in-gel electrophoresis. We analysed high-quality eggs from domesticated and wild pikeperch fish to reveal proteins that were presumably only related to the domestication process and not to the quality of eggs. Here, we show that domestication has a profound impact on the protein profile of pikeperch eggs. We identified 66 differentially abundant protein spots, including 27 spots that were more abundant in wild-caught pikeperch eggs and 39 spots that were enriched in eggs collected from domesticated females. Eggs originating from wild-caught females showed higher expression levels of proteins involved in folding, apoptotic process, purine metabolism and immune response, whereas eggs of domesticated females showed higher expression levels of proteins that participated mainly in metabolism. The changes in metabolic proteins in eggs from domesticated females can reflect the adaptation of pikeperch to commercial diets, which have profoundly distinct compositions compared with natural diets. The decrease in the abundance of proteins related to immune response in eggs from the domesticated population suggests that domestication may lead to disturbances in defence mechanisms. In turn, the lower abundance of heat shock proteins in eggs of domesticated fish may indicate their adaptation to stable farming conditions and reduced environmental stressors or their better tolerance of stress from breeding. The proteins identified in this study can increase our knowledge concerning the mechanism of the pikeperch domestication process.     

Proteome characterization of the unsequenced psychrophile Pedobacter cryoconitis using 15N metabolic labeling, tandem mass spectrometry, and a new bioinformatic workflow

Organisms without a sequenced genome and lacking a complete protein database encounter an added level of complexity to protein identification and quantitation. De novo sequencing, new bioinformatics tools, and mass spectrometry (MS) techniques allow for advances in this area. Here, the proteomic characterization of an unsequenced psychrophilic bacterium, Pedobacter cryoconitis, is presented employing a novel workflow based on (15) N metabolic labelling, 2DE, MS/MS, and bioinformatics tools. Two bioinformatics pipelines, based on nitrogen constraint (N-constraint), ortholog searching, and de novo peptide sequencing with N-constraint similarity database search, are compared based on proteome coverage and throughput. Results demonstrate the effect of different growth temperatures (1°C, 20°C) and different carbon sources (glucose, maltose) on the proteome. Seventy-six and 69 proteins were identified and validated from the glucose- and maltose-grown bacterium, respectively, from which 21 and 22 were differentially expressed at different growth temperatures. Differentially expressed proteins are involved in stress response and carbohydrate metabolism, with higher expression at 20°C than at 1°C, while antioxidants were upregulated at 1°C. This study provides an alternative workflow to identify, validate, and quantify proteins from unsequenced organisms distantly related to other species in the protein database. Furthermore, it provides further understanding on bacterial adaptation mechanisms to cold environments, and a comparative proteomic analyses with other psychrophilic microorganisms.     

Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress

Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions.     

Discovery,characterization, and remediation of a C-terminal Fc-extension in proteins expressed in CHO cells

ABSTRACT

Protein-based biotherapeutics are produced in engineered cells through complex processes and may contain a wide variety of variants and post-translational modifications that must be monitored or controlled to ensure product quality. Recently, a low level (~1–5%) impurity was observed in a number of proteins derived from stably transfected Chinese hamster ovary (CHO) cells using mass spectrometry. These molecules include antibodies and Fc fusion proteins where Fc is on the C-terminus of the construct. By liquid chromatography-mass spectrometry (LC-MS), the impurity was found to be ~1177 Da larger than the expected mass. After tryptic digestion and analysis by LC-MS/MS, the impurity was localized to the C-terminus of Fc in the form of an Fc sequence extension. Targeted higher-energy collision dissociation was performed using various normalized collision energies (NCE) on two charge states of the extended peptide, resulting in nearly complete fragment ion coverage. The amino acid sequence, SLSLSPEAEAASASELFQ, obtained by the de novo sequencing effort matches a portion of the vector sequence used in the transfection of the CHO cells, specifically in the promoter region of the selection cassette downstream of the protein coding sequence. The modification was the result of an unexpected splicing event, caused by the resemblance of the commonly used GGU codon of the C-terminal glycine to a consensus splicing donor. Three alternative codons for glycine were tested to alleviate the modification, and all were found to completely eliminate the undesirable C-terminal extension, thus improving product quality.     

Identification and disruption of btlA,a locus involved in bile tolerance and general stress resistance in Listeria monocytogenes

A transposon Tn917 mutant of Listeria monocytogenes L028 was isolated on the basis of reduced growth on agar adjusted to pH 5.5. The disrupted gene, designated btlA (bile tolerance locus), encodes a putative secondary transporter of the major facilitator superfamily, which has significant homology to yxiO in Bacillus subtilis (lmo1417 in L. monocytogenes EGDe). The mutant demonstrated decreased growth rates relative to the wild-type when grown in sub-lethal levels of various stressors (acid, salt, ethanol, bile, SDS, ampicillin and phosphomycin). The mutant was also more sensitive to lethal levels of bile. A pORI19 insertion mutant demonstrated similar phenotypes. Murine virulence studies indicated that disruption of btlA does not influence virulence potential. BtlA therefore represents a membrane protein essential for the maintenance of homeostasis under stress conditions, but is not involved in pathogenicity.     

Proteomic analysis of mouse jejunal epithelium and its response to infection with the intestinal nematode, Trichinella spiralis

Infection with the intestinal nematode Trichinella spiralis induces profound, but stereotypic pathological changes to the epithelium, which are common to many nematode infections. This study describes changes in jejunal epithelial protein expression that reflect these stereotypic responses. Adult male BALB/c mice were infected with T. spiralis, and groups (n = 4) examined on day 14/15 (time of worm rejection) were compared with uninfected controls (n = 4). Jejunal epithelium was harvested and extracted for two-dimensional gel electrophoresis. Tryptic peptide mass fingerprinting was used to create a reference map consisting of a total of 52 landmark spots. Of these, 16 were observed to change in intensity during infection. The changes observed at day 14/15 were of relevance to such mechanisms as lipid utilization and transport (increase in triacylglycerol lipase, and reduction in intestinal fatty acid binding protein) and innate immunity (appearance of intelectin-2). As a result, candidate molecules have been identified for further focused studies on their role in the host response to intestinal nematode infection.     

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.     

Targeted label-free quantitative analysis of secretory proteins from adipocytes in response to oxidative stress

Adipocytes are well known to release regulation factors associated with metabolic disorders. In particular, increased oxidative stress in adipocytes contributes to dysregulation of adipokine production. In this study, we applied relative quantitative proteomic analysis based on label-free multiple reaction monitoring (MRM) to discover biological changes of adipokines under oxidative stress. Among a total of 194 identified proteins, 8 proteins were selected and quantified between control and hydrogen peroxide (H₂O₂)-treated groups by label-free MRM quantification. The secretion levels of matrix metalloproteinase-2 (MMP-2), stromal cell-derived factor-1 (SDF-1, CXCL12), resistin, and complement factor D (CFD, adipsin) decreased, whereas the secretion levels of tissue inhibitor of metalloproteinase-2 (TIMP-2) and aldolase A increased. Here we suggest that our study with label-free quantitative analysis will contribute to the efficient quantitative analysis of targeted proteins in complex mixtures and specifically to a better understanding of changes of adipokines under oxidative stress.     

Differential expression of proteins in response to the interaction between the pathogen Fusarium graminearum and its host, Hordeum vulgare

Using proteomic techniques, a study aimed at isolating and identifying proteins associated with resistance to fusarium head blight (FHB) was conducted on six barley genotypes of varying resistance. At anthesis, barley spikelets were point inoculated with Fusarium graminearum macroconidial suspensions or mock inoculum. In total, 43 acidic protein spots out of 600 were detected 3 days postinoculation to be differentially expressed due to FHB and were identified. Identification of proteins responsive to FHB included those associated with oxidative burst and oxidative stress response, such as malate dehydrogenase and peroxidases, and pathogenesis-related (PR). An increase in abundance of PR-3 or PR-5 could be associated with the resistant genotypes CI4196, Svansota, and Harbin, as well as the intermediate resistant genotype CDC Bold. On the contrary, the susceptible genotype Stander showed a decrease in abundance of these acidic PR-proteins. In the susceptible and intermediate resistant genotypes Stander and CDC Bold, as well as CI4196, the increased abundance of proteins associated with an oxidative response might have prepared the terrain for saprophytic fungal invasion. On the contrary, in the resistant sources Harbin and Svansota we did not observed change in abundance of these proteins. Not a single significant change in acidic protein abundance could be detected in Chevron. Three distinct response patterns are reported from these six barley genotypes.     

饥饿胁迫下家蚕5龄起蚕中的蛋白表达谱及 BmLp-c 6的转录分析

【目的】分析家蚕Bombyx mori受饥饿胁迫后的蛋白质谱变化,探索其耐受饥饿的机理。【方法】以家蚕品种932为实验材料,利用双向电泳和质谱技术检测5龄起蚕经过24 h饥饿胁迫的蛋白质谱差异变化,利用荧光定量PCR技术分析BmLp-c 6的转录表达。【结果】经比对饥饿蚕和正常取食蚕的血淋巴蛋白谱,饥饿蚕有62个特异蛋白点。蛋白点的等电点在4.22~6.98之间,分子量分布在20.81～144.69 kDa间。选取只在饥饿时出现的特异蛋白点No. 7111进行质谱鉴定,根据其氨基酸序列进行引物设计,获得了目的基因BmLp-c 6,经与载体pET-21d(+)连接重组后,成功获得诱导表达。经实时荧光定量PCR分析,当5龄起蚕受到饥饿胁迫影响时,BmLp-c 6基因在血淋巴中大量转录表达,但在中肠中的转录表达水平却极低。【结论】家蚕5龄起蚕在饥饿胁迫下,血淋巴中的蛋白质谱发生变化,BmLp-c 6会大量转录表达。     

Macrophage-capping protein as a tissue biomarker for prediction of response to gemcitabine treatment and prognosis in cholangiocarcinoma

Cholangiocarcinoma is one of the deadliest malignancies worldwide. Recent studies reported that treatment with gemcitabine was effective in prolonging survival. However, as the treatment only benefited a limited subset of patients, selection of patients before treatment is required. To discover biomarkers predictive of the response to gemcitabine treatment in cholangiocarcinoma, we examined the proteome of three types of material resource; ten cell lines, nine xenografts and nine surgically resected primary tumors from patients who exhibited different response to gemcitabine treatment. Two-dimensional difference gel electrophoresis generated quantitative protein expression profiles including 3571 protein spots. We detected 172 protein spots with significant correlation with response to gemcitabine treatment. All proteins corresponding to these 172 protein spots were identified by mass spectrometry. We found that the macrophage-capping protein (CapG) was associated with response to gemcitabin treatment in all three types of material source. Immunohistochemical validation in an additional set of 196 cholangiocarcinoma cases revealed that CapG expression was associated with lymphatic invasion status and overall survival. Multivariate analysis showed that CapG protein expression was an independent prognostic factor for overall survival. In conclusion, CapG was identified as a novel candidate biomarker to predict response to gemcitabine treatment and survival in cholangiocarcinoma.