OFFGEL Electrophoresis Research Articles

Protective Effects of an Octapeptide Identified from Riceberry™ (Oryza sativa) Protein Hydrolysate on Oxidative and Endoplasmic Reticulum (ER) Stress in L929 Cells.

Reactive oxygen species (ROS) play a critical role in oxidative stress and cellular damage, underscoring the importance of identifying potent antioxidants. This research focuses on the antioxidant capabilities of Riceberry™-derived peptides and their protective effects against oxidative and endoplasmic reticulum (ER) stress in L929 cells. By simulating human digestion, Riceberry™ protein hydrolysate was generated, from which antioxidant peptides were isolated using OFFGEL electrophoresis and LC-MS/MS. Notably, an octapeptide (VPAGVAHW) from the hydrolysate demonstrated significant antioxidant activity, particularly against oxidative stress induced by iodoacetic acid (IAA) or hydrogen peroxide (H2O2) and ER stress caused by tunicamycin (TM) in L929 cells. This peptide's effectiveness was evident in its dose-dependent ability to enhance cell viability and mitigate stress effects, although its efficiency varied with the stress inducer. Our study suggests that Riceberry™-derived peptides could serve as a promising natural antioxidant with potential benefits for health promotion and applications in the food industry, offering an environmentally friendly alternative to synthetic antioxidants.

Optimization of Novel GELFrEE Fractionation for Molecular Weight–Based In-solution Protein Separation from Buffalo Meat, Pork, and Chicken

This study evaluates and compares the novel GELFrEE protein fractionation technique with OFFGEL electrophoresis in their performance as in-solution fractionation approaches for total and myofibrillar/sarcoplasmic proteins isolated from different food animals and poultry meat. The current study demonstrates that both OFFGEL and GELFrEE techniques are suitable for separation of relatively low-abundant proteins with an average of 0.125 pI width or 1.0 kDa resolution and effectively reduce the sample complexity in 24–36 h and 2–3 h, respectively. The 5%, 8%, and 12% GEFREE 8100 cartridges are effective in fractionating total buffalo meat proteins within a mass range of 10–240 kDa, 10–140 kDa, and 5–45 kDa, respectively. Our work also provides the usefulness of GELFrEE separation with an average fraction width of 40 kDa (5%), 10 kDa (8%), and 4.0 kDa (12%) for further complementary protein identification using MALDI-TOF MS. We have demonstrated a maximum protein loading capacity of 2.5 mg and 8.0 mg for OFFGEL and GELFrEE with up to 50–100% sample recovery for the latter. The performed comparison of the fractionation techniques demonstrates the potential of GELFrEE fractionator for rapid, reproducible, high degree of resolution and high sample recovery tool in proteomic applications.

In-depth proteomic characterization of Schistosoma haematobium: Towards the development of new tools for elimination.

BackgroundSchistosomiasis is a neglected disease affecting hundreds of millions worldwide. Of the three main species affecting humans, Schistosoma haematobium is the most common, and is the leading cause of urogenital schistosomiasis. S. haematobium infection can cause different urogenital clinical complications, particularly in the bladder, and furthermore, this parasite has been strongly linked with squamous cell carcinoma. A comprehensive analysis of the molecular composition of its different proteomes will contribute to developing new tools against this devastating disease.Methods and findingsBy combining a comprehensive protein fractionation approach consisting of OFFGEL electrophoresis with high-throughput mass spectrometry, we have performed the first in-depth characterisation of the different discrete proteomes of S. haematobium that are predicted to interact with human host tissues, including the secreted and tegumental proteomes of adult flukes and secreted and soluble egg proteomes. A total of 662, 239, 210 and 138 proteins were found in the adult tegument, adult secreted, soluble egg and secreted egg proteomes, respectively. In addition, we probed these distinct proteomes with urine to assess urinary antibody responses from naturally infected human subjects with different infection intensities, and identified adult fluke secreted and tegument extracts as being the best predictors of infection.ConclusionWe provide a comprehensive dataset of proteins from the adult and egg stages of S. haematobium and highlight their utility as diagnostic markers of infection intensity. Protein composition was markedly different between the different extracts, highlighting the distinct subsets of proteins that different development stages present in their different niches. Furthermore, we have identified adult fluke ES and tegument extracts as best predictors of infection using urine antibodies of naturally infected people. This study provides the first steps towards the development of novel tools to control this important neglected tropical disease.

Investigation of the global protein content from healthy human tears

Considering absence of invasiveness and side effects, tears emerge as a particularly attractive fluid for biomarker discovery and therefore for daily clinical use. However, to date this fluid remains poorly studied in healthy condition. Here, we present an updated in-depth characterisation of the human healthy tear protein composition using proteomics approach. Both eyes of 8 healthy controls (4 men and 4 women, average age: 38 ± 18) were collected using the Schirmer's strip method. After liquid digestion and off-gel electrophoresis fractionation, three independent proteomics analyses were performed on a LTQ-Orbitrap Velos Pro. Globally, 1351 proteins were identified with 2 unique peptides and 1% FDR. Gene ontology analyses showed up that 39% of the tear proteins were enzymes, with high numbers of dehydrogenases, phosphatases, kinases and ligases. Immunoglobulins, serpins and 14-3-3 domains proteins also emerged as the main tear protein families. The glycolysis and the coagulation and complement cascades, which were already shown in tears as involved in ocular and systemic diseases, were highlighted performing pathway analyses. Our study therefore complements the existing data on healthy tears proteome. Nevertheless, extensive studies for deeply and definitively characterise this promising fluid are required in the near future in order to be able to routinely use this fluid in clinics. A better understanding of its protein content will probably open new avenues in the biomarker discovery and clinical practice in the near future.

Deep Dive on the Proteome of Human Cerebrospinal Fluid: A Valuable Data Resource for Biomarker Discovery and Missing Protein Identification.

Cerebrospinal fluid (CSF) is a body fluid of choice for biomarker studies of brain disorders but remains relatively under-studied compared with other biological fluids such as plasma, partly due to the more invasive means of its sample collection. The present study establishes an in-depth CSF proteome through the analysis of a unique CSF sample from a pool of donors. After immunoaffinity depletion, the CSF sample was fractionated using off-gel electrophoresis and analyzed with liquid chromatography tandem mass spectrometry (MS) using the latest generation of hybrid Orbitrap mass spectrometers. The shotgun proteomic analysis allowed the identification of 20 689 peptides mapping on 3379 proteins. To the best of our knowledge, the obtained data set constitutes the largest CSF proteome published so far. Among the CSF proteins identified, 34% correspond to genes whose transcripts are highly expressed in brain according to the Human Protein Atlas. The principal Alzheimer's disease biomarkers (e.g., tau protein, amyloid-β, apolipoprotein E, and neurogranin) were detected. Importantly, our data set significantly contributes to the Chromosome-centric Human Proteome Project (C-HPP), and 12 proteins considered as missing are proposed for validation in accordance with the HPP guidelines. Of these 12 proteins, 8 proteins are based on 2 to 6 uniquely mapping peptides from this CSF analysis, and 4 match a new peptide with a "stranded" single peptide in PeptideAtlas from previous CSF studies. The MS proteomic data are available to the ProteomeXchange Consortium ( http://www.proteomexchange.org/ ) with the data set identifier PXD009646.

Identification of Missing Proteins in Normal Human Cerebrospinal Fluid.

The cerebrospinal fluid (CSF) proteome data set presented herein was obtained after immunodepletion of abundant proteins and off-gel electrophoresis fractionation of a commercial pool of normal human CSF; liquid chromatography tandem mass spectrometry analysis was performed with a linear ion trap-Orbitrap Elite. We report the identification of 12 344 peptides mapping on 2281 proteins. In the context of the Chromosome-centric Human Proteome Project (C-HPP), the existence of seven missing proteins is proposed to be validated. This data set is available to the ProteomeXchange Consortium ( http://www.proteomexchange.org/ ) with the data set identifier PXD008029.

Revisiting the Ancylostoma Caninum Secretome Provides New Information on Hookworm-Host Interactions.

Hookworm infection is a major tropical parasitic disease affecting almost 500 million people worldwide. These soil-transmitted helminths can survive for many years in the intestine of the host, where they feed on blood, causing iron deficiency anemia and other complications. These parasites release a variety of molecules known as excretory/secretory products (ESPs) that are involved in many different biological processes that govern parasite survival. Using a combination of separation techniques such as SDS-PAGE and OFFGEL electrophoresis, in combination with state-of-the-art mass spectrometry we have reanalyzed the dog hookworm, Ancylostoma caninum, ESPs (AcESP). We identified 315 proteins present in the AcESP, compared with just 105 identified in previous studies. The most highly represented family of proteins is the SCP/TAPs (110 of the 315 proteins), and the most abundant constituents of AcESP are homologues of the tissue inhibitors of metalloproteases (TIMP) family. Interestingly, we identified new homologs of well-known vaccine candidates and immunomodulatory proteins. This study provides novel information about the proteins secreted by A. caninum, and constitutes a comprehensive dataset to study the proteins involved in host-hookworm interactions.

Study on the CFHR1 level and its genetic polymorphisms in type 2 diabetes mellitus patients

To explore the characteristics in CFHR1 concentration and the frequency of CFHR1 gene polymorphisms of patients with type 2 diabetes mellitus (T2DM) based on the high level of complement factor H (CFH) expression among such patients and the similarity between CFHR1 and CFH in terms of sequence and functions. Fifty T2DM patients and 30 healthy controls were selected. The plasma samples were separated by pI with OFFGEL electrophoresis following solution digestion. Further separation and identification were carried out on a Nano HPLC-Chip-MS/MS system. Differentially expressed proteins were identified by comparison. Enzyme-linked immunosorbent assay (ELISA) was used to validate the result. Genomic DNA of the two groups was extracted. Polymerase chain reaction and sequencing were used to determine the single nucleotide polymorphisms in the 6 exons of the CFHR1 gene. The CFHR1 level in plasma of T2DM patients were significantly higher than that of the healthy controls (P=2.78× 10-11). A significant difference in allelic frequencies of rs12406079 of the fifth exon of the CFHR1 gene was found between the two groups (χ 2=5.692, P=0.017). The concentration of CFHR1 and frequencies of CFHR1 gene polymorphisms among patients with T2DM differ significantly from healthy subjects. Polymorphisms of the CFHR1 gene are associated with T2DM.

Quantitative Shotgun Proteomics Unveils Candidate Novel Esophageal Adenocarcinoma (EAC)-specific Proteins

Esophageal cancer is the eighth most common cancer worldwide and the majority of patients have systemic disease at presentation. Esophageal adenocarcinoma (OAC), the predominant subtype in western countries, is largely resistant to current chemotherapy regimens. Selective markers are needed to enhance clinical staging and to allow targeted therapies yet there are minimal proteomic data on this cancer type. After histological review, lysates from OAC and matched normal esophageal and gastric samples from seven patients were subjected to LC MS/MS after tandem mass tag labeling and OFFGEL fractionation. Patient matched samples of OAC, normal esophagus, normal stomach, lymph node metastases and uninvolved lymph nodes were used from an additional 115 patients for verification of expression by immunohistochemistry (IHC).Over six thousand proteins were identified and quantified across samples. Quantitative reproducibility was excellent between technical replicates and a moderate correlation was seen across samples with the same histology. The quantitative accuracy was verified across the dynamic range for seven proteins by immunohistochemistry (IHC) on the originating tissues. Multiple novel tumor-specific candidates are proposed and EPCAM was verified by IHC.This shotgun proteomic study of OAC used a comparative quantitative approach to reveal proteins highly expressed in specific tissue types. Novel tumor-specific proteins are proposed and EPCAM was demonstrated to be specifically overexpressed in primary tumors and lymph node metastases compared with surrounding normal tissues. This candidate and others proposed in this study could be developed as tumor-specific targets for novel clinical staging and therapeutic approaches.

OFFGEL electrophoresis and tandem mass spectrometry approach compared with DNA-based PCR method for authentication of meat species from raw and cooked ground meat mixtures containing cattle meat, water buffalo meat and sheep meat

The present study compared the accuracy of an OFFGEL electrophoresis and tandem mass spectrometry-based proteomic approach with a DNA-based method for meat species identification from raw and cooked ground meat mixes containing cattle, water buffalo and sheep meat. The proteomic approach involved the separation of myofibrillar proteins using OFFGEL electrophoresis, SDS-PAGE and protein identification by MALDI-TOF MS. Species-specific peptides derived from myosin light chain-1 and 2 were identified for authenticating buffalo meat spiked at a minimum 0.5% level in sheep meat with high confidence. Relative quantification of buffalo meat mixed with sheep meat was done by quantitative label-free mass spectrometry using UPLC-QTOF and PLGS search engine to substantiate the confidence level of the data. In the DNA-based method, PCR amplification of mitochondrial D loop gene using species specific primers found 226bp and 126bp product amplicons for buffalo and cattle meat, respectively. The method was efficient in detecting a minimum of 0.5% and 1.0% when buffalo meat was spiked with cattle meat in raw and cooked meat mixes.

Comparison of bioactive peptides prepared from sheep cheese whey using a food‐grade bacterial and a fungal protease preparation

SummaryNovel bacterial (HT) and fungal (FPII) food‐grade protease preparations were evaluated for their ability to hydrolyse sheep cheese whey (SCW) and the generation of bioactive peptides. Both protease preparations hydrolysed the whey proteins to small peptides over 24‐h hydrolysis time, but the time course hydrolysis profiles were different as evaluated by SDS‐PAGE. The HT whey hydrolysate had considerably higher antioxidant and angiotensin‐I converting enzyme (ACE)‐inhibitor activity than the FPII hydrolysate. Neither hydrolysate was cytotoxic towards Vero cells. OFFGEL electrophoresis of the small peptide pool fraction (<15 amino acids) of each hydrolysate indicated differences in the pI distribution of the bioactive peptides. This likely reflects the diverse hydrolytic specificity of the proteases. Although the antioxidant activity of both hydrolysates was not significantly affected by simulated gastrointestinal digestion, the loss of ACE‐inhibitor activity was greater with the FPII hydrolysate.

Proteomic based approach for characterizing 4-hydroxy-2-nonenal induced oxidation of buffalo (Bubalus bubalis) and goat (Capra hircus) meat myoglobins.

BackgroundMyoglobin (Mb) is a sarcoplasmic heme protein primarily responsible for meat color and its chemistry is species specific. 4-hydroxy-2-nonenal (HNE) is a cytotoxic lipid derived aldehyde detected in meat and was reported to covalently adduct with nucleophilic histidine residues of Mb and predispose it to greater oxidation. However, no literature is available on characterization of lipid oxidation induced oxidation of Indian water buffalo (Bubalus bubalis) and goat (Capra hircus) myoglobins.MethodsPresent study characterize the Mb extracted from water buffalo and goat cardiac muscles using two-dimensional gel electrophoresis (2DE), OFFGEL electrophoresis and mass spectrometry (MS). Purified buffalo and goat bright red oxymyoglobin were reacted with HNE in-vitro at physiological pH (7.4) and temperature (37 °C) conditions and the formation of oxidised brown metmyoglobin was measured. The Mb-HNE adducts were detected using MALDI-TOF MS, whereas specific sites of adduction was determined using ESI-QTOF MS/MS.ResultsPurified buffalo and goat Mb samples revealed a molecular mass of 17,043.6 and 16,899.9 Daltons, respectively. The 2DE analysis exhibited 65 (sarcoplasmic protein extract) and 6 (pure Mb) differentially expressed (P < 0.05) protein spots between buffalo and goat samples. OFFGEL electrophoresis revealed an isoelectric point of 6.77 and 7.35 respectively, for buffalo and goat Mb’s. In-vitro incubation of HNE with bright red buffalo and goat oxymyoglobin’s at pH 7.4 and 37 °C resulted in pronounced (P < 0.05) oxidation and formation of brown metmyoglobin. MALDI-TOF MS analysis of Mb-HNE reaction mix revealed covalent binding (via Michael addition) of 3 and 5 molecules of HNE with buffalo and goat Oxy-Mb’s, respectively. ESI-QTOF MS/MS identified seven and nine histidine (HIS) residues of Mb that were readily adducted by HNE in buffalo and goat, respectively.ConclusionThe study demonstrated better redox stability of buffalo Mb than goat Mb. Our findings confirm the hypothesis that relative effect of HNE was greater for Mb’s with 12 ± 1 HIS residues than Mb’s with 9 HIS residues and helps meat processors in developing species-specific processing strategies to reduce the color variability.

Exploration of human tear proteome

PurposeThe tear film is a complex structure which constitutes an interface between our eyes and the external environment. Its protein, lipid and metabolite composition is highly regulated depending on several factors. Despite its attractive characteristics, this fluid is still poorly studied. Here we present an in‐depth human tear proteome based on a mass spectrometry approach.MethodsIn this study, tears of two healthy controls (two women aged 59 and 61 years) were collected with Schirmer's papers. After trypsin digestion and off‐gel electrophoresis fractionation, two proteomic analyses were performed by mass spectrometry (using LTQ Orbitrap Velos Pro coupled to a liquid chromatography). Resulting files were searched against the UniProt‐SwissProt/TrEMBL database (version 2014_10) and a false discovery rate of 1% was selected. The protein list was analysed using Ingenuity Pathways Analysis and Cytoscape software.ResultsGlobally 2105 and 825 proteins were identified with 1 and 2 unique peptides respectively after removing keratins and immunoglobulins. Regarding the 825 proteins, the top three pathways that we highlighted were the acute phase response signalling, the remodelling of epithelial adherens junctions and the clathrin‐mediated endocytosis signalling. Moreover, we identified 203 proteins that were not found in the previous published studies. By comparing our tears with others fluids, only 26.9% were identified in vitreous humor and 45.7% in plasma, confirming that tears have a specific composition.ConclusionsThanks to this study, we are able to propose an expanded tear proteome. Both specific proteins of the tears and correlations with other fluids give them a great potential for biomarker research.This study is kindly supported by the Provisu Foundation and the SNF_MVH (PMPDP3_158370).

Purification of plant complex protein extracts in non-denaturing conditions by in-solution isoelectric focusing

An alternative approach for plant complex protein extracts pre-purification by in-solution isoelectric focusing in non-denaturing conditions is presented. The separation of biologically active proteins, in narrow ranges of isoelectric point (pI) was obtained by a modified OFFGEL electrophoresis. Two different water-soluble protein extracts from Phragmites leaves were fractionated into 24 fractions within a 3–10 pI range at 10 °C in the absence of denaturing/reducing agents. One-dimensional electrophoretic analysis revealed different protein distribution patterns and the effective fractionation of both protein extracts. Peroxidase activity of each fraction confirmed that proteins remained active and pre-purification occurred. Biological triplicates assured the needed reproducibility.

Towards generation of bioactive peptides from meat industry waste proteins: Generation of peptides using commercial microbial proteases

Five commercially available food-grade microbial protease preparations were evaluated for their ability to hydrolyse meat myofibrillar and connective tissue protein extracts to produce bioactive peptides. A bacterial-derived protease (HT) extensively hydrolysed both meat protein extracts, producing peptide hydrolysates with significant in vitro antioxidant and ACE inhibitor activities. The hydrolysates retained bioactivity after simulated gastrointestinal hydrolysis challenge. Gel permeation chromatography sub-fractionation of the crude protein hydrolysates showed that the smaller peptide fractions exhibited the highest antioxidant and ACE inhibitor activities. OFFGEL electrophoresis of the small peptides of both hydrolysates showed that low isoelectric point peptides had antioxidant activity; however, no consistent relationship was observed between isoelectric point and ACE inhibition. Cell-based assays indicated that the hydrolysates present no significant cytotoxicity towards Vero cells. The results indicate that HT protease hydrolysis of meat myofibrillar and connective tissue protein extracts produces bioactive peptides that are non-cytotoxic, should be stable in the gastrointestinal tract and may contain novel bioactive peptide sequences.

Dissolved proteins characterization and speciation studies of metal-protein complexes in marine sediment pore water

Dissolved proteins characterization in marine sediment pore water has been performed by offgel electrophoresis (OGE) and microfluidic lab-on-chip (LOC) electrophoresis, assessing for the first time dissolved protein characteristics such as isoelectric point (pIs), and molecular weight (MW). In addition, speciation studies of metal-protein complexes in sediment pore water have also been performed by direct analysis of offgel fractions by electrothermal atomic absorption spectrometry (ETAAS), and inductively coupled plasma optical emission spectrometry (ICP-OES). Samples were subjected to a tangential flow ultrafiltration (UF) followed by a centrifugal UF (10kDa molecular weight cut-off, MWCO) procedure, which offered a pre-concentration factor of 450. Isolation of dissolved proteins from the extracts has been performed by treating the ultrafiltrated fractions with methanol-chloroform as a precipitating reagent. The protein pellets were dried, dissolved in the offgel buffer, and subjected to optimized OGE and LOC electrophoresis procedures. Results showed proteins of MWs ranging from 5.1 to 87.0kDa, and proteins exhibiting the highest MW were found in offgel fractions comprising pIs within the 6.34–7.76 range. Regarding metal-protein complexes, cadmium (Cd), and lead (Pb) were not found in any of the analyzed offgel fractions, which implies low affinity of these elements to the isolated proteins. However, iron (Fe) was found to be associated to proteins with pIs between 3.0 and 10 (the highest Fe concentrations were found in proteins from offgel fractions with pIs 6.3 to 7.7). Finally, copper (Cu), manganese (Mn), and zinc (Zn) were found to be bound to proteins exhibiting low MWs.

Porous silica enhanced proteolysis during Off-Gel separation for efficient protein identification

An advanced approach is developed in this work for simultaneous on-line separation and digestion of proteins by combining the Off-Gel isoelectric focusing (IEF) and enzymatic nanoreactor enhanced proteolysis. The nanoreactor was prepared by preloading trypsin in amino-functionalized macroporous silica, and then directly added into Off-Gel wells. With the nanoreactor loaded Off-Gel device, effective digestion of proteins happened during IEF electrophoresis to generate directly fractionated tryptic peptides, which not only accelerated the experimental flow but also avoided sample loss, leading to a more comprehensive protein identification from complex biological samples. A successful identification of 3592 proteins was achieved from Hela cell line by using the approach followed with LC–MS/MS analysis, while only 1877 proteins were identified from the same sample when using standard in-solution proteolysis followed with Off-Gel electrophoresis and then LC–MS/MS analysis. Therefore, we have demonstrated that the approach can greatly simplify high-throughput proteomics and significantly improve protein identification.

Quantitative changes in proteins responsible for flavonoid and anthocyanin biosynthesis in strawberry fruit at different ripening stages: A targeted quantitative proteomic investigation employing multiple reaction monitoring.

To gain insight into the molecular mechanisms contributing to flavonoids and anthocyanin biosynthesis and regulation of strawberry fruit during ripening is challenging due to limited molecular biology tools and established hypothesis. Our targeted proteomic approach employing LC-MS/MS analysis and MRM technique to quantify proteins in relation to flavonoids and anthocyanin biosynthesis and regulation in strawberry fruit during fruit ripening is novel. The identification of peptides and proteins provided reliable design and validation of quantitative approaches using SRM on targeted proteins proposed involved in strawberry fruit. Our data revealed the identifying candidate proteins and their quantitative changes in relation to fruit ripening and flavonoids and anthocyanin biosynthesis and regulation. More importantly, this quantitative proteomic data is also compared with chemical analysis to reveal possible control levels of this important quality trait. Although, MRM approach is not new in plant biology research, the application has been very rare. This is the first systematic multi-targeted interrogation of the possible regulation of entire pathway of flavonoids and anthocyanin biosynthesis in strawberry fruit at different ripening stages using quantitative MRM technique on mass spectrometry. Our results demonstrate the power of targeted quantitative mass spectrometry data for analysis of proteins in biological regulation. These results indicate that distinct and diverse control of flavonoids and anthocyanin biosynthesis mechanisms at metabolism and proteins levels. This important and complementary knowledge will be useful for systematically characterizing the flavonoids and anthocyanin biosynthesis pathway of any fruit/plant species.

Evaluation of offgel electrophoresis, electrothermal atomic absorption spectroscopy and inductively coupled plasma optical emission spectroscopy for trace metal analysis in marine plankton protein

Two-dimensional offgel and lab-on-a-chip (LOC) electrophoresis was performed for assessing proteins from marine plankton. Proteins were isolated from plankton samples by using a phenol/sodium dodecylsulfate (SDS) buffer (2:1) pH8.0 mixture as an extracting solution. The SDS buffer consisted of 30% (m/v) sucrose, 2% (m/v) SDS, 0.1M of Tris–HCl, pH8.0, 5% (v/v) β-mercaptoethanol and protease inhibitor cocktail. Before protein extraction, the plankton were subjected to different washing steps involving 10% trichloroacetic acid (TCA)/acetone and methanol. Proteins exhibiting the highest molecular weights (MWs), within the 52.6–57.3kDa range, were found in offgel fractions of isoelectric points (pIs) ranging from 4.40 to 8.60; whereas, the highest protein concentrations were found in fraction 5 (100μgg−1 for a protein of pI of 4.5 and MW of 22.0kDa), fraction 16 (55μgg−1, protein of pI/MW of 7.4/19.1kDa), and fraction 8 (51μgg−1, pI/MW of 5.3/22.2kDa). Further studies were also developed for determining the amount of trace metals associated with the isolated proteins of similar pIs by electrothermal atomic absorption spectroscopy (ETAAS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Preliminary results indicate that Fe, Ni, Mn and Zn were found to be associated with proteins of variable MW (from 14 to 54kDa) and pIs (from 3.4 to 8.2). Otherwise, Cd was found to be associated with proteins of certain pIs (6.0–6.5) and with MWs ranging from 14.1 to 53.1kDa.

Proteomic analysis of Synechocystis sp. PCC6803 responses to low-temperature and high light conditions

The global changes in protein expression of Synechocystis sp. PCC6803, a photosynthetic bacterium for the production of secondary metabolites as a green cell factory, were investigated by proteome separation and a subsequent tandem mass spectrometry. Two different proteome separation techniques, strong cation exchange chromatography and off-gel electrophoresis, were applied. The combination of the two proteome separation techniques enabled the comparative analysis of the differential regulation of the Synechocystis proteome in response to two different environmental factors, temperature and light. A total of 1,483 proteins were identified, which represent over 40% of the genes in Synechocystis. Our data showed that fatty acid metabolism was inhibited by (3R)-hydroxymyristol acyl carrier protein dehydrase (Sll1605) under low temperature conditions. The expression of UDP-N-acetylglucosamine acyltransferase (Sll0379) and 3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase (Slr0776), which is involved in lipopolysaccharide metabolism, was not observed under high light conditions. Under high light exposure, proteins related to iron-sulfur metabolism were detected, which may be responsible for maintaining the redox potential of the photosystem. High light under low temperature caused severe damage to the photosystem. Some of the responses to these stresses were similar to those previously reported for other photosynthetic organisms. Notably, this study revealed the followings: (i) low temperature inhibits fatty acid synthesis; (ii) high light inhibits lipopolysaccharides synthesis and stimulates the expression of iron-sulfur related proteins; and (iii) high light under low temperature induces the photorespiratory cycle. The global proteomic analysis clearly showed that stress conditions such as low temperature and/or high light induce cellular metabolisms related with the protection of their photosystems in the model microalga Synechocystis sp. PCC6803.