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In contrast to bottom-up LC-MS only 2DE-MS can separate and detect a huge number of human protein species. Kwiatkowski et al. (in this issue) established parameters to estimate the amount of protein speciation for each human protein. Proteins identified in 2DE-MS approaches showed more protein speciation than in bottom-up LC-MS. The authors state that protein speciation is likely to increase the chance of proteins to be determined in 2-DE/MS, though admitting that low-sensitivity 2DE-MS methods were used in this study. In agreement with Kwiatkowski et al., we are convinced that the difference between 2DE-MS and bottom-up LC-MS will disappear, if high-resolution 2DE is combined with identification by a high-sensitivity LC-Orbitrap-MS. Meta-analysis of proteomic data is surely a promising tool, though the technological progress in 2DE and MS has to reach a plateau to enable useful comparisons.

The proteasome is a large polymeric protease complex responsible for degradation of intracellular proteins and generation of peptides. In this study, we purified a native 20S proteasome protein complex from zebrafish (

Phosphate-binding tag (Phos-tag) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is an important development capable of analyzing the phosphorylation state of proteins. Conventionally, proteins were separated via SDS-PAGE and Phos-tag SDS-PAGE that use different gels to identify phosphorylated proteins. However, it was often difficult to compare the electrophoretic mobility of the proteins in the different gels used. The recently developed Phos-tag diagonal electrophoresis has been able to solve this problem. It can indicate the SDS-PAGE and Phos-tag SDS-PAGE patterns on a single gel; therefore, phosphorylated proteins can be distinguished easily from non-phosphorylated proteins. This review assesses the importance of Phos-tag electrophoresis, which enables the analysis of protein phosphorylation states, in the field of proteomics. Additionally, this review describes the significance and actual experimental technique of Phos-tag diagonal electrophoresis, which was recently developed to overcome the drawbacks of Phos-tag SDS-PAGE. Although shotgun analysis of proteins allows detecting many phosphorylation sites, it is challenging to clarify the differences in the phosphorylation states of protein molecules using this technique. Therefore, Phos-tag SDS-PAGE is frequently used to determine the phosphorylation state of proteins. This technique has become more powerful with the recent development of Phos-tag diagonal electrophoresis. Abbreviations: BIS, N,N’-methylenebis(acrylamide); CBB, Coomassie brilliant blue R250; ESI, electrospray ionization; hnRNP, heterogeneous ribonucleoprotein K; LTQ–Orbitrap, Linear trap quadrupole–Orbitrap; LC, liquid chromatography; MS, mass spectrometry; MALDI, matrix-assisted laser desorption ionization; Phos-tag, phosphate-binding tag [1,3-bis [bis (pyridine-2-ylmethyl) amino] propane-2-olate]; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TOF, time of flight; 2D-DIGE, fluorescence-labeled two-dimensional difference gel electrophoresis; 2-DE, two-dimensional gel electrophoresis

Human sparganosis is a food-borne parasitic disease caused by the plerocercoids of Spirometra species. Clinical diagnosis of sparganosis is crucial for effective treatment, thus it is important to identify sensitive and specific antigens of plerocercoids. The aim of the current study was to identify and characterize the immunogenic proteins of Spirometra erinaceieuropaei plerocercoids that were recognized by patient sera. Crude soluble extract of the plerocercoids were separated using 2-dimensional gel electrophoresis coupled with immunoblot and mass spectrometry analysis. Based on immunoblotting patterns and mass spectrometry results, 8 antigenic proteins were identified from the plerocercoid. Among the proteins, cysteine protease protein might be developed as an antigen for diagnosis of sparganosis.

Proteome-pI 2.0 is an update of an online database containing predicted isoelectric points and pKa dissociation constants of proteins and peptides. The isoelectric point—the pH at which a particular molecule carries no net electrical charge—is an important parameter for many analytical biochemistry and proteomics techniques. Additionally, it can be obtained directly from the pKa values of individual charged residues of the protein. The Proteome-pI 2.0 database includes data for over 61 million protein sequences from 20 115 proteomes (three to four times more than the previous release). The isoelectric point for proteins is predicted by 21 methods, whereas pKa values are inferred by one method. To facilitate bottom-up proteomics analysis, individual proteomes were digested in silico with the five most commonly used proteases (trypsin, chymotrypsin, trypsin + LysC, LysN, ArgC), and the peptides’ isoelectric point and molecular weights were calculated. The database enables the retrieval of virtual 2D-PAGE plots and customized fractions of a proteome based on the isoelectric point and molecular weight. In addition, isoelectric points for proteins in NCBI non-redundant (nr), UniProt, SwissProt, and Protein Data Bank are available in both CSV and FASTA formats. The database can be accessed at http://isoelectricpointdb2.org., Graphical Abstract Graphical AbstractProteome-pI 2.0 database: 61,329,034 proteins from 20,115 proteomes with isoelectric point predicted using 21 methods; 5.38B dissociation constant (pKa) predictions; 9.58B in silico digested peptides (Trypsin, Chymotrypsin, Trypsin+LysC, LysN, ArgC).

Several years have passed since LC (liquid chromatography)-MS (mass spectrometry) became the mainstream for proteomic analysis; however, conventional fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) continues to be an important technology that enables rapid and direct visualization of hundreds to thousands of proteins and their quantitative analyses. We can get global proteomic views using 2D-DIGE within 3 days and then identify proteins with differential expression levels using MALDI-TOF/MS and MASCOT search engine. Here, we describe our routine 2D-DIGE proteomic analysis of the liver isolated from mice in pathological conditions within 1 week.

Cancer of blood or bone marrow-derived cells dysregulates normal hematopoiesis and accounts for over 6% of all cancer cases annually. Proteomic analyses of blood cancers have improved our understanding of disease mechanisms and identified numerous proteins of clinical relevance. For many years, gel-based proteomic studies have aided in the discovery of novel diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, in various diseases, including blood cancer. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) facilitates comparative proteomic research to identify differential protein expression in a simple and reproducible manner. The versatility of 2D-DIGE as a quantitative proteomic technique has provided insight into various aspects of blood cancer pathology, including disease development, prognostic subtypes, and drug resistance. The ability to couple 2D-DIGE with additional downstream mass spectrometry-based techniques yields comprehensive workflows capable of identifying proteins of biological and clinical significance. The application of 2D-DIGE in blood cancer research has significantly contributed to the increasingly important initiative of precision medicine. This chapter will focus on the influential role of 2D-DIGE as a tool in blood cancer research.

Here, we describe a detailed step-by-step protocol for the detection of phosphoproteins in two-dimensional difference gel electrophoresis (2D-DIGE) gels. A standard 2D-DIGE protocol is combined with subsequent post-staining with phosphospecific fluorescent dye. The combination of these two methods complements 2D-DIGE-based proteome profiling by fluorescence detection of phosphoproteins in the same gel providing additional possibility for sensitive and accurate quantification of the differentially regulated phosphoproteins in biological samples.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation, and accurate within-gel matching. 2D-DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS (sodium dodecyl sulfate)-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometric techniques and further analyzed by other biochemical methods. In this chapter, we describe application of this methodology to separation and quantitation of protein mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in meat industry.

The skeletal muscle proteome consists of a large number of diverse protein species with a broad and dynamic concentration range. Since mature skeletal muscles are characterized by a distinctive combination of contractile cells with differing physiological and biochemical properties, it is essential to determine specific differences in the protein composition of fast, slow, and hybrid fibers. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a powerful comparative tool to analyze fiber type-specific differences between predominantly fast contracting versus slower twitching muscles. In this chapter, the application of the 2D-DIGE method for the comparative analysis of different subtypes of skeletal muscles is outlined in detail. A standardized proteomic workflow is described, involving sample preparation, protein extraction, differential fluorescence labeling using a 3-CyDye system, first-dimension isoelectric focusing, second-dimension slab gel electrophoresis, 2D-DIGE image analysis, protein digestion, and mass spectrometry.

Enzyme activity assay methods can be used to corroborate the results generated by difference gel electrophoresis (DIGE) proteomic experiments. Two assay methods were chosen to demonstrate how this can be achieved. Assays for determining the activity of superoxide dismutase and NADH dehydrogenase are outlined in detail in this chapter. These methods were chosen as examples because they are frequently used in conjunction with DIGE proteomics.

The discovery of clinically relevant biomarkers using gel-based proteomics has proven extremely challenging, principally because of the large dynamic range of protein abundances in biofluids such as blood and the fact that only a small number of proteins constitute the vast majority of total blood protein mass. Various separation, depletion, enrichment, and quantitative developments coupled with improvements in gel-based protein quantification technologies, specifically fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), have contributed to significant improvements in the detection and identification of lower abundance proteins. One of these enrichment technologies, ProteoMiner, is the focus of this chapter. The ProteoMiner technology utilizes hexapeptide bead library with huge diversity to bind and enrich low-abundance proteins but at the same time suppresses the concentration of high-abundance proteins in subsequent analysis.

The gradual loss of skeletal muscle mass during aging and associated decline in contractile strength can result in reduced fitness, frailty, and loss of independence. In order to better understand the molecular and cellular mechanisms that underlie sarcopenia of old age and the frailty syndrome, as well as identify novel therapeutic targets to treat age-related fiber wasting, it is crucial to develop a comprehensive biomarker signature of muscle aging. Fluorescence two-dimensional gel electrophoresis (2D-DIGE) in combination with sensitive mass spectrometry presents an ideal bioanalytical tool for biomarker discovery in biogerontology. This chapter outlines the application of the 2D-DIGE method for the comparative analysis of human biopsy specimens from middle-aged versus senescent individuals using a two-CyDye-based method.

Two-dimensional difference gel electrophoresis (2D-DIGE) is a high-resolution protein separation technique, with the excellent dynamic range obtained by fluorescent tag labeling of protein samples. Scanned images of 2D-DIGE gels show thousands of protein spots, each spot representing a single or a group of protein isoforms. By using commercially available software, each protein spot is defined by an outline, which is digitized and correlated with the quantity of proteins present in each spot. Software packages include DeCyder, SameSpots, and Dymension 3. In addition, proteins of interest can be excised from post-stained gels and identified with conventional mass spectrometric techniques. High-throughput mass spectrometry is performed using sophisticated instrumentation, including matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), MALDI-TOF/TOF, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Tandem MS (MALDI-TOF/TOF or LC-MS/MS) analyzes fragmented peptides, resulting in amino acid sequence information, which is especially useful when protein spots are low abundant or where a mixture of proteins is present.

The global analysis of the proteome is an important tool in cell biology. Comparative proteomic evaluations can identify and compare the composition, dynamics, and modifications between different samples. Comparing tissue proteomes under different conditions is crucial for advancing the biomedical field. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a sensitive and robust biochemical method that can compare multiple protein samples over a broad dynamic range on the same analytical gel and can be used to establish differentially expressed protein profiles between different sample groups. 2D-DIGE involves fluorescently labeling protein samples with CyDye flours, via a two-dye or a three-dye system, pre-separation by isoelectric point, and molecular weight. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization, thus enabling accurate high-resolution analysis of differences in protein abundance between samples. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of two-dye and three-dye DIGE minimal labeling.

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a key biochemical method for the comparative analysis of complex protein mixtures. The technique focuses on the identification and characterization of individual protein species following gel electrophoretic separation making it an important analytical tool of top-down proteomics. In order to verify changes in the expression levels of a particular protein, as determined by 2D-DIGE analysis, and evaluate the subcellular localization of the proteoform of interest, immunofluorescence microscopy is very well suited. This chapter describes in detail the preparation of tissue specimens and the process of cryo-sectioning, as well as incubation with primary antibodies and fluorescently labeled secondary antibodies, followed by image analysis. As illustrative examples, the co-detection of immuno-labeled dystrophin and the Y-chromosome in skeletal muscle are shown, and the localization of calbindin in the cerebellum is presented.

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a widely employed method for efficient protein separation and the determination of abundance changes in distinct proteoforms. This makes this gel-based method a key technique of comparative approaches in top-down proteomics. For the appropriate screening of proteome-wide alterations, initial preparative steps involve sample handling, homogenization, subcellular fractionation, and the determination of protein concentration, which makes the optimal application of these techniques a crucial part of a successful initiation of a new 2D-DIGE-based analysis. This chapter describes sample homogenization and a standardized protein assay for the preparation of homogenates with a known protein concentration for subsequent differential fluorescent tagging and two-dimensional gel electrophoretic separation.

The combination of large-scale protein separation techniques, sophisticated mass spectrometry, and systems bioinformatics has led to the establishment of proteomics as a distinct discipline within the wider field of protein biochemistry. Both discovery proteomics and targeted proteomics are widely used in biological and biomedical research, whereby the analytical approaches can be broadly divided into proteoform-centric top-down proteomics versus peptide-centric bottom-up proteomics. This chapter outlines the scientific value of top-down proteomics and describes how fluorescence two-dimensional difference gel electrophoresis can be combined with the systematic analysis of crucial post-translational modifications. The concept of on-membrane digestion following the electrophoretic transfer of proteins and the usefulness of comparative two-dimensional immunoblotting are discussed.

Proteins can be separated according to their size by gel electrophoresis and further analyzed by Western blotting. The proteins can be transferred to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF), which results in a replica of the protein's separation patterns. The proteins on the membrane can be detected by specific antibodies followed by visualization either on the membrane itself, on film, or by CCD cameras. Western blotting is a sensitive technique to verify data obtained from fluorescence two-dimensional difference gel electrophoresis (2D-DIGE)-based proteomics.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an elegant gel electrophoretic analytical tool for comparative protein assessment. It is based on two-dimensional gel electrophoresis (2D-GE) separation of fluorescently labeled protein extracts. The tagging procedures are designed to not interfere with the chemical properties of proteins with respect to their pI and electrophoretic mobility, once a proper labeling protocol is followed. The use of an internal pooled standard makes 2D-DIGE a highly accurate quantitative method enabling multiple protein samples to be separated on the same two-dimensional gel. Technical limitations of this technique (i.e., underrating of low abundant, high molecular mass and integral membrane proteins) are counterbalanced by the incomparable separation power which allows proteoforms and unknown PTM (posttranslational modification) identification. Moreover, the image matching and cross-gel statistical analysis generates robust quantitative results making data validation by independent technologies successful.

Assays for measuring enzyme activity can be useful tools for proteomics applications. Enzyme testing can be performed to validate an experimental system prior to a Difference Gel Electrophoresis (DIGE) proteomic experiment and can also be utilized as an integral part of multifaceted experiment in conjunction with DIGE. Data from enzyme tests can be used to corroborate results of DIGE proteomic experiments where an enzyme or enzymes are demonstrated by DIGE to be differentially expressed. Enzyme testing can also be utilized to support data from DIGE experiments that demonstrate metabolic changes in a biological system. The different types of enzyme assays that can be performed in conjunction with DIGE experiments are reviewed alongside a discussion of experimental approaches for designing enzyme assays.

The biochemical and cell biological profiling of contractile fiber types and subcellular structures plays a central role in basic and applied myology. Mass spectrometry-based proteomics presents an ideal approach for the systematic identification of proteomic and subproteomic markers. These representative components of fast versus slow muscle fibers and their subcellular fractions are highly useful for in-depth surveys of skeletal muscle adaptations to physiological challenges, as well as the improvement of diagnostic, prognostic, and therapy-monitoring methodologies in muscle pathology. This chapter outlines the identification of subproteomic markers for skeletal muscle profiling based on bottom-up and top-down approaches, including fluorescence two-dimensional difference gel electrophoresis (2D-DIGE).

In-gel digestion of protein spots derived from two-dimensional gels and their subsequent identification by mass spectrometry is involved in a multitude of mass spectrometry-driven proteomic experiments, including fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). This type of proteomic methodology has been involved in the establishment of comparative proteome maps and in the identification of differentially expressed proteins and their isoforms in health and disease. Most in-gel digestion protocols follow a number of common steps including excision of the protein spots of interest, destaining, reduction and alkylation (for silver-stained gels), and dehydration and overnight digestion with the proteolytic enzyme of choice. While trypsin has been a mainstay of peptide digestion for many years, it does have its shortcomings, particularly related to incomplete peptide digestion, and this has led to a rise in popularity for other proteolytic enzymes either used alone or in combination. This chapter discusses the alternative enzymes available and describes the process of in-gel digestion using the enzyme trypsin.

Many biomedically relevant biomarkers are proteins with characteristic biochemical properties and a relatively restricted subcellular distribution. The comparative and mass spectrometry-based proteomic analysis of body fluids can be particularly instrumental for the targeted identification of novel protein biomarkers with pathological relevance. In this respect, new research efforts in biomarker discovery focus on the systematic mapping of the human saliva proteome, as well as the pathobiochemical identification of disease-related modifications or concentration changes in specific saliva proteins. As a product of exocrine secretion, saliva can be considered an ideal source for the biochemical identification of new disease indicators. Importantly, saliva represents a body fluid that is continuously available for diagnostic and prognostic assessments. This chapter gives an overview of saliva proteomics, including a discussion of the usefulness of both liquid chromatography and two-dimensional gel electrophoresis for efficient protein separation in saliva proteomics.

Identification of target molecules of new bioactive compounds is still a challenge in drug development. Various proteomics-based methods have been developed to analyze the interaction between compounds and target proteins. Among these methods, cellular thermal shift assay (CETSA) has been frequently applied in recent years for validation studies of compound-protein interactions using antibodies. Combining CETSA with comprehensive proteomic analysis has been successful in narrowing down the target(s) of a new compound from the enormous number of proteins in cell. In this chapter, we introduce 2DE-CETSA, which combines CETSA with proteome analysis using two-dimensional electrophoresis as a method for identification of target proteins.

The use of tumor markers aids in the early detection of cancer recurrence and prognosis. There is a hope that they might also be useful in screening tests for the early detection of cancer. Here, the question of finding ideal tumor markers, which should be sensitive, specific, and reliable, is an acute issue. Human plasma is one of the most popular samples as it is commonly collected in the clinic and provides noninvasive, rapid analysis for any type of disease including cancer. Many efforts have been applied in searching for “ideal” tumor markers, digging very deep into plasma proteomes. The situation in this area can be improved in two ways—by attempting to find an ideal single tumor marker or by generating panels of different markers. In both cases, proteomics certainly plays a major role. There is a line of evidence that the most abundant, so-called “classical plasma proteins”, may be used to generate a tumor biomarker profile. To be comprehensive these profiles should have information not only about protein levels but also proteoform distribution for each protein. Initially, the profile of these proteins in norm should be generated. In our work, we collected bibliographic information about the connection of cancers with levels of “classical plasma proteins”. Additionally, we presented the proteoform profiles (2DE patterns) of these proteins in norm generated by two-dimensional electrophoresis with mass spectrometry and immunodetection. As a next step, similar profiles representing protein perturbations in plasma produced in the case of different cancers will be generated. Additionally, based on this information, different test systems can be developed.

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in

Natural-based compounds with repellent activity arise nowadays with the possibility to replace commercial synthetic repellents wholly or partially, such as N,N-Diethyl-m-toluamide (DEET). It is due to DEET's demonstrated toxicity and cutaneous irritation for human beings. Besides, research recommends avoiding using it with kids and pregnant women. The search for a repellent product implies early stages of detailed research that resolve the modes of action against the target insect. Therefore the objective of the current study was to analyze neuronal electrophysiological signals and olfactory system protein expression when the Aedes aegypti mosquito with exposition to natural-based repellents. Adult females of Ae. aegypti of Rockefeller strain were exposed to specific concentrations of repellent compounds like geranyl acetate, α-bisabolol, nerolidol, and DEET. The neuronal effect was measured by electroantennography technique, and the effect of exposure to either DEET or a mixture of natural molecules on protein expression was determined with 2D-PAGE followed by MALDI-TOF-mass spectrometry (MS). This approach revealed that DEET affected proteins related to synapses and ATP production, whereas natural-based repellents increased transport, signaling, and detoxification proteins. The proteomic and electrophysiology experiments demonstrated that repellent exposure disrupts ionic channel activity and modifies neuronal synapse and energy production processes.

Uterine cancers are among the most prevalent gynecological malignancies, and endometrial cancer (EC) is the most common in this group. This study used tissue-based proteomic profiling analysis in patients with endometrial cancer and hyperplasia, and control patients. Conventional 2D gel electrophoresis, followed by a mass spectrometry approach with bioinformatics, including a network pathway analysis pipeline, was used to identify differentially expressed proteins and associated metabolic pathways between the study groups. Thirty-six patients (twelve with endometrial cancer, twelve with hyperplasia, and twelve controls) were enrolled in this study. The mean age of the participants was 46–75 years. Eighty-seven proteins were significantly differentially expressed between the study groups, of which fifty-three were significantly differentially regulated (twenty-eight upregulated and twenty-five downregulated) in the tissue samples of EC patients compared to the control (Ctrl). Furthermore, 26 proteins were significantly dysregulated (8 upregulated and 18 downregulated) in tissue samples of hyperplasia (HY) patients compared to Ctrl. Thirty-two proteins (nineteen upregulated and thirteen downregulated) including desmin, peptidyl prolyl cis-trans isomerase A, and zinc finger protein 844 were downregulated in the EC group compared to the HY group. Additionally, fructose bisphosphate aldolase A, alpha enolase, and keratin type 1 cytoskeletal 10 were upregulated in the EC group compared to those in the HY group. The proteins identified in this study were known to regulate cellular processes (36%), followed by biological regulation (16%). Ingenuity pathway analysis found that proteins that are differentially expressed between EC and HY are linked to AKT, ACTA2, and other signaling pathways. The panels of protein markers identified in this study could be used as potential biomarkers for distinguishing between EC and HY and early diagnosis and progression of EC from hyperplasia and normal patients.

Pasteurella multocida is an important zoonotic pathogen that causes multiple diseases in both animals and humans. Test of good immunogenic proteins is beneficial for vaccine development and disease control. In the present study, we determined four novel immunogenic proteins of P. multocida by using 2-DE MALDI-TOF MS with immune serum. These four proteins included a trimethylamine-N-oxide reductase TorA, a translation elongation factor Ts, a phosphoglyceromutase PGAM, and a peroxiredoxin PrX. Among these proteins, TorA, Prx, and PGAM were successfully expressed by using E. coli. Western-blotting assays showed that recombinant TorA, Prx, and/or PGAM displayed good reactions with infectious sera of P. multocida serogroups A, B, D and F. Immunization of either rTorA, rPrx, and/or rPGAM induced significantly high levels of antibodies as well as IFN-γ, IL-4 and IL-10 in mice (P 0.01). Protective efficacy tests revealed that vaccination of either rTorA, rPrx, and/or rPGAM protected 60% ~ 80% of the tested mice against the challenge with P. multocida field isolate. Our results obtained from the present study suggest that these three proteins could be tested as good vaccine candidates against P. multocida infections.

Background:Aside from its pervasiveness, whereby it affects as much as 20% of the world's population, depression continues to be one of the most crucial psychiatric problems due to the loss of power it causes by disrupting daily life functioning, containing economic consequences, and having a high suicidal tendency. Major depression (MD) is a systemic and multifactorial disorder involving complex interactions between genetic predisposition and disturbances of various molecular pathways.Objectives:In our current study, we aimed to identify the proteins obtained from serum samples that change during depression with the MD model.Methods:The MD model was applied through the forced swim test in rats. 14 Winstar Albino male rats were divided into two equal groups as follows: depression and control groups. Serum samples were separated by chromatographic methods and then compared with two-dimensional (2D) electrophoresis.Results:A total of 9 potential diagnostic protein sequences were identified, which were distinguished with computer software. During the last phase of the study, the Matrix-Assisted Laser Desorption/ Ionization – Time of Flight (MALDI-TOF) analysis, the previous expression sequences identified among the groups were determined and classified. By comparing protein expressions, it was concluded that 9 different points could be used together as a potential biomarker.Conclusion:Results can help us identify a new diagnostic system that can be used to diagnose MD.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific.

Comparative proteomic profiling of human follicular fluid (HFF) from polycystic ovary syndrome (PCOS) and non-PCOS patients who displayed low levels of fertility was carried out via two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry. HFF, an important reproductive fluid, was used for the proteomic analysis of PCOS patients to determine the effect of PCOS on folliculogenesis. HFF was obtained from 10 women (average age, 35 years) undergoing in vitro fertilization at two hospitals. The proteins of HFF were separated using 2-DE analysis and validated by Western blot assay. Approximately 250 protein spots were separated on the 2-DE gel. Among them, the expression levels of seven proteins were found to change at least 1.5-fold in the PCOS patient group. Three protein spots, albumin, uncharacterized protein 1, and uncharacterized protein 2, were downregulated in PCOS patients. However, four protein spots, gelsolin, vitamin D binding protein, serum albumin, and complement factor B, were upregulated in PCOS patient group. These proteins may serve as a panel of potential pathological biomarkers during fertilization and oocyte development.

Two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry were performed to compare the proteomic alterations of lycorine-treated and control cells to further investigate the anti-multiple myeloma (MM) mechanisms of lycorine. Mass spectrometry results showed that after lycorine treatment of MM cells, 42% of the differentially expressed proteins had subcellular localization, mainly, on mitochondria. Voltage-dependent anion-selective channel protein 2 (VDAC2), the most abundant protein in the outer mitochondrial membrane, was up-regulated after treatment with lycorine and was subsequently verified by western blot analysis. Further studies on mitochondria found that lycorine was able to increase abnormal mitochondria and increase mitochondrial membrane potential. Lycorine can achieve the effect of resisting multiple myeloma by acting on VDAC2 and causing mitochondrial abnormalities.

We present the first proteomic analysis on the cellular response to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. The differential proteomes of Vero E6 cells with and without infection of the SARS-CoV were resolved and quantitated with two-dimensional differential gel electrophoresis followed by ESI-MS/MS identification. Moreover isotope-coded affinity tag technology coupled with two-dimensional LC-MS/MS were also applied to the differential proteins of infected cells. By combining these two complementary strategies, 355 unique proteins were identified and quantitated with 186 of them differentially expressed (at least 1.5-fold quantitative alteration) between infected and uninfected Vero E6 cells. The implication for cellular responses to virus infection was analyzed in depth according to the proteomic results. Thus, the present work provides large scale protein-related information to investigate the mechanism of SARS-CoV infection and pathogenesis.

Neurodegenerative diseases are caused by progressive lesions or loss of specific nerve cells, which endanger human health. However, the mechanism by which neurodegeneration manifests remains unclear. Proteomics can shed light on this question as well as help establish diagnostic standards and discover new drug targets. The power of proteomics for understanding neurodegenerative diseases has increased substantially with the application of iTRAQ and TMT labeling techniques. This review focuses on progress in these labeling techniques and their applications in neurodegeneration research.

The specific cytotoxic effects of nanoparticles on tumor cells may be used in future antitumor clinical applications. Gold nanoparticles (AuNPs) have been reported to produce potent cytotoxic effects; however, the precise mechanism is unclear. In this study, AuNPs were synthesized; the average size of the particles was 62.2 ± 6 nm with smooth surface and multiple shapes, which were determined using transmission electron microscopy and field emission scanning electron microscopy. The selected area electron diffraction patterns suggested that the synthesized AuNPs were crystalline. The X-ray photoelectron spectroscopy (XPS) spectrum of the synthesized AuNPs has presented an intense peak at 100 eV, signifying the entire composition of Au in the developed AuNPs. This synthesized AuNPs showed the most potent efficacy in prostate cancer cells, regardless of whether or not they were androgen dependent. Secretome determinations using two-dimensional difference in-gel electrophoresis (2D-DIGE), followed by enzyme-linked immunosorbent assay and quantitative reverse transcriptase-polymerase chain reaction validations, have identified a series of secretory proteins that were dysregulated by AuNP treatment in prostate cancer cells, many of which are highly involved in cytokine-chemokine functions, including CXCL3, interleukin-10, CCL2, and matrix metalloproteinase 9 (MMP9). Further research on molecular mechanism has indicated that AuNPs can trigger the secretion of anticancer factors and myeloid cell-polarizing factors from tumor cells through MMP9 inhibition. These results have clearly signified the cytotoxic potential of AuNPs for treating prostate cancer and may provide a novel direction for prostate cancer therapy in the future.