There is a great diversity of protein samples types and origins, therefore the optimal procedure for each sample type must be determined empirically. In order to obtain a reproducible and complete sample presentation which view as many proteins as possible on the desired 2DE gel, it is critical to perform additional sample preparation steps to improve the quality of the final results, yet without selectively losing the proteins. To address this, we developed a general method that is suitable for diverse sample types based on phenolchloroform extraction method (represented by TRI reagent). This method was found to yield good results when used to analyze human breast cancer cell line (MCF-7), Vibrio cholerae, Cryptocaryon irritans cyst and liver abscess fat tissue. These types represent cell line, bacteria, parasite cyst and pus respectively. For each type of samples, several attempts were made to methodically compare protein isolation methods using TRI-reagent Kit, EasyBlue Kit, PRO-PREP™ Protein Extraction Solution and lysis buffer. The most useful protocol allows the extraction and separation of a wide diversity of protein samples that is reproducible among repeated experiments. Our results demonstrated that the modified TRI-reagent Kit had the highest protein yield as well as the greatest number of total proteins spots count for all type of samples. Distinctive differences in spot patterns were also observed in the 2DE gel of different extraction methods used for each type of sample.
The use of lectin affinity chromatography prior to 2-DE separation forms an alternative method to unmask the expression of targeted glycoproteins of lower abundance in serum samples. Reduced expression of alpha-2 macroglobulin (AMG) and complement factor B (CFB) was detected in sera of patients with nasopharyngeal carcinoma (NPC) when pooled serum samples of the patients and those of healthy individuals were subjected to affinity isolation using immobilized champedak mannose-binding lectin and analyzed by 2-DE and densitometry. The AMG and CFB spots were not detected in the 2-DE protein profiles when the same pooled serum samples were subjected to albumin and IgG depletion and neither were they detected when the depleted samples were analyzed by western blotting and lectin detection. Together with other acute-phase response proteins that were previously reported to be altered in expression in NPC patients, AMG and CFB may serve as useful complementary biomarkers for NPC.
The typical concentration of protein loaded varies from 0.13 to 1.40 μg/μL for a classical silver staining method in 2DE gel. Here, we present a simple modified classical silver staining method by modifying the silver impregnation and development reaction steps. This modified method detects the protein spots at extremely low loaded concentrations, ranging from 0.0048 to 0.0480 μg/μL. We recommend this modified silver staining as an excellent method for the limited biological samples used for silver-stained 2DE analysis. Altogether, the protocol takes close to two days from first dimension separation to second dimension separation, followed by silver staining, scanning, and analysis.
Sera of IgA nephropathy (IgAN) patients and normal subjects were analysed by two-dimensional (2-D) gel electrophoresis. Densitometric analysis of the 2-D gels of IgAN patients and normal subjects revealed that their protein maps were comparable. There was no shift of pI values in the major alpha-heavy chain spots. However, the volume of the alpha-heavy chain bands were differently distributed. Distribution was significantly lower at the anionic region in IgAN patients (mean anionic:cationic ratio of 1.184 +/- 0.311) as compared to normal healthy controls (mean anionic:cationic ratio of 2.139 +/- 0.538). Our data are in support of the previously reported findings that IgA1 of IgAN patients were lacking in sialic acid residues.
Ganoderma species are a group of fungi that have the ability to degrade lignin polymers and cause severe diseases such as stem and root rot and can infect economically important plants and perennial crops such as oil palm, especially in tropical countries such as Malaysia. Unfortunately, very little is known about the complex interplay between oil palm and Ganoderma in the pathogenesis of the diseases. Proteomic technologies are simple yet powerful tools in comparing protein profile and have been widely used to study plant-fungus interaction. A critical step to perform a good proteome research is to establish a method that gives the best quality and a wide coverage of total proteins. Despite the availability of various protein extraction protocols from pathogenic fungi in the literature, no single extraction method was found suitable for all types of pathogenic fungi. To develop an optimized protein extraction protocol for 2-DE gel analysis of Ganoderma spp., three previously reported protein extraction protocols were compared: trichloroacetic acid, sucrose and phenol/ammonium acetate in methanol. The third method was found to give the most reproducible gels and highest protein concentration. Using the later method, a total of 10 protein spots (5 from each species) were successfully identified. Hence, the results from this study propose phenol/ammonium acetate in methanol as the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp.
Oil palm (Elaeis guineensis) is an important economic crop cultivated for its nutritional palm oil. A significant amount of effort has been undertaken to understand oil palm growth and physiology at the molecular level, particularly in genomics and transcriptomics. Recently, proteomics studies have begun to garner interest. However, this effort is impeded by technical challenges. Plant sample preparation for proteomics analysis is plagued with technical challenges due to the presence of polysaccharides, secondary metabolites and other interfering compounds. Although protein extraction methods for plant tissues exist, none work universally on all sample types. Therefore, this study aims to compare and optimize different protein extraction protocols for use with two-dimensional gel electrophoresis of young and mature leaves from the oil palm. Four protein extraction methods were evaluated: phenol-guanidine isothiocyanate, trichloroacetic acid-acetone precipitation, sucrose and trichloroacetic acid-acetone-phenol. Of these four protocols, the trichloroacetic acid-acetone-phenol method was found to give the highest resolution and most reproducible gel. The results from this study can be used in sample preparations of oil palm tissue for proteomics work.
Researchers frequently use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D-PAGE method, which 'inverts' the dimension of protein separation of the conventional 2D-PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first-dimension separation was conducted on a gel-free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second-dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D-PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D-PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D-PAGE. Overall, the i2D-PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.
Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen.
Palm oil is an edible vegetable oil derived from lipid-rich fleshy mesocarp tissue of oil palm (Elaeis guineensis Jacq.) fruit and is of global economic and nutritional relevance. While the understanding of oil biosynthesis in plants is improving, the fundamentals of oil biosynthesis in oil palm still require further investigations. To gain insight into the systemic mechanisms that govern oil synthesis during oil palm fruit ripening, the proteomics approach combining gel-based electrophoresis and mass spectrometry was used to profile protein changes and classify the patterns of protein accumulation during these complex physiological processes. Protein profiles from different stages of fruit ripening at 10, 12, 14, 15, 16, 18 and 20 weeks after anthesis (WAA) were analysed by two-dimensional gel electrophoresis (2DE). The proteome data were then visualised using a multivariate statistical analysis of principal component analysis (PCA) to get an overview of the proteome changes during the development of oil palm mesocarp. A total of 68 differentially expressed protein spots were successfully identified by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF/TOF) and functionally classified using ontology analysis. Proteins related to lipid production, energy, secondary metabolites and amino acid metabolism are the most significantly changed proteins during fruit development representing potential candidates for oil yield improvement endeavors. Data are available via ProteomeXchange with identifier PXD009579. This study provides important proteome information for protein regulation during oil palm fruit ripening and oil synthesis.
The primary components of human hair shaft-keratin and keratin-associated proteins (KAPs), together with their cross-linked networks-are the underlying reason for its rigid structure. It is therefore requisite to overcome the obstacle of hair insolubility and establish a reliable protocol for the proteome analysis of this accessible specimen. The present study employed an alkaline-based method for the efficient isolation of hair proteins and subsequently examined them using gel-based proteomics. The introduction of two proteomic protocols, namely the conventional and modified protocol, have resulted in the detection of more than 400 protein spots on the two-dimensional gel electrophoresis (2DE). When compared, the modified protocol is deemed to improve overall reproducibility, whilst offering a quick overview of the total protein distribution of hair. The development of this high-performance protocol is hoped to provide a new approach for hair analysis, which could possibly lead to the discovery of biomarkers for hair in health and diseases in the future.
Cancers can cause some proteins to be aberrantly excreted or released in the urine, which can be used as biomarkers. To screen for potential biomarkers for endometrial cancer (ECa), the urinary proteins from patients who were newly diagnosed with early stage ECa and untreated controls were separated using two-dimensional gel electrophoresis (2-DE) and followed by image analysis. The altered levels of zinc alpha-2 glycoprotein, alpha 1-acid glycoprotein, and CD59 were detected in the patients compared to the controls. In addition, the urine of the ECa patients was also found to contain relatively lower levels of a fragment of nebulin when the 2-DE separated urinary proteins were probed using champedak galactose binding (CGB) lectin. The different levels of the nebulin fragment were further validated by subjecting the urinary protein samples to CGB lectin affinity chromatography and analysis of the bound fractions by LC-MS/MS. Our data is suggestive of the potential use of the differentially expressed urinary proteins as biomarkers for ECa although this requires further extensive validation on clinically representative populations.
Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method.
Familial hypercholesterolaemia (FH) and Familial defective apolipoprotein B100 (FDB) are autosomal dominant inherited diseases of lipid metabolism caused by mutations in the low density lipoprotein (LDL) receptor and apolipoprotein B 100 genes. FH is clinically characterised by elevated concentrations of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C), presence of xanthomata and premature atherosclerosis. Both conditions are associated with coronary artery disease but may be clinically indistinguishable. Seventy-two (72) FH patients were diagnosed based on the Simon Broome's criteria. Mutational screening was performed by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Positive mutations were subjected to DNA sequencing for confirmation of the mutation. We successfully amplified all exons in the LDL receptor and apo B100 genes. DGGE was performed in all exons of the LDL receptor (except for exons 4-3', 18 and promoter region) and apo B100 genes. We have identified four different mutations in the LDL receptor gene but no mutation was detected in the apo B 100 gene. The apo B100 gene mutation was not detected on DGGE screening as sequencing was not performed for negative cases on DGGE technique. To our knowledge, the C234S mutation (exon 5) is a novel mutation worldwide. The D69N mutation (exon 3) has been reported locally while the R385W (exon 9) and R716G (exon 15) mutations have not been reported locally. However, only 4 mutations have been identified among 14/72 patients (19.4%) in 39 FH families. Specificity (1-false positive) of this technique was 44.7% based on the fact that 42/76 (55.3%) samples with band shifts showed normal DNA sequencing results. A more sensitive method needs to be addressed in future studies in order to fully characterise the LDLR and apo B100 genes such as denaturing high performance liquid chromatography. In conclusion, we have developed the DNA analysis for FH patients using PCR-DGGE technique. DNA analysis plays an important role to characterise the type of mutations and forms an adjunct to clinical diagnosis.
Vitamin E has been suggested to modulate age-associated changes by altering the redox balance resulting in altered gene and/or protein expression. Here we have utilized proteomics to determine whether such regulation in protein expression occurs in human lymphocytes from two different age groups stressed with H₂O₂ and then treated with vitamin E in the form of tocotrienol-rich fraction (TRF). In this study, lymphocytes obtained from young (30-49 years old) and old (>50 years old) volunteers were first challenged with 1 mM H₂O₂. They were then treated by exposure to 50, 100 and 200 μg/ml TRF. Two-dimensional gel electrophoresis followed by MALDI-TOF/TOF (matrix-assisted laser desorption/ionization time-of-flight/time-of-flight) tandem mass spectrometry was then performed on whole-cell protein extracts to identify proteins that have changed in expression. A total of 24 proteins were found to be affected by H₂O₂ and/or TRF treatment. These included proteins that were related to metabolism, antioxidants, structural proteins, protein degradation and signal transduction. Of particular interest was the regulation of a number of proteins involved in stress response--peroxiredoxin-2, peroxiredoxin-3 and peroxiredoxin-6-all of which were shown to be down-regulated with H₂O₂ exposure. The effect was reversed following TRF treatment. The expression of peroxiredoxin-2 and peroxiredoxin-6 was confirmed by quantitative reverse transcriptase polymerase chain reaction. These results suggested that TRF directly influenced the expression dynamics of the peroxiredoxin-2, thus improving the cells ability to resist damage caused by oxidative stress.
Four different methods were evaluated to extract proteins from "Musang King" durian pulps and subsequently proteins with different abundance between fresh and long term frozen storage were identified using two-dimensional polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analyses. The acetone-phenol method was found to produce good protein yields and gave the highest gel resolution and reproducibility. Differential protein analyses of the durian pulp revealed that 15 proteins were down-regulated and three other proteins were up-regulated after a year of frozen storage. Isoflavone reductase-like protein, S-adenosyl methionine synthase, and cysteine synthase isoform were up-regulated during frozen storage. The down-regulation of proteins in frozen durian pulps indicated that frozen storage has affected proteins in many ways, especially in their functions related to carbohydrate and energy metabolisms, cellular components, and transport processes. This study will enable future detailed investigations of proteins associated with quality attributes of durians to be studied.
Although cervical cancer is preventable with early detection, it remains the second most common malignancy among women. An understanding of how proteins change in their expression during a particular diseased state such as cervical cancer will contribute to an understanding of how the disease develops and progresses. Potentially, it may also lead to the ability to predict the occurrence of the disease. With this in mind, we aimed to identify differentially expressed proteins in the plasma of cervical cancer patients. Plasma from control, cervical intraepithelial neoplasia (CIN) grade 3 and squamous cell carcinoma (SCC) stage IV subjects was resolved by two-dimensional gel electrophoresis and the resulting proteome profiles compared. Differentially expressed protein spots were then identified by mass spectrometry. Eighteen proteins were found to be differentially expressed in the plasma of CIN 3 and SCC stage IV samples when compared with that of controls. Competitive ELISA further validated the expression of cytokeratin 19 and tetranectin. Functional analyses of these differentially expressed proteins will provide further insight into their potential role(s) in cervical cancer-specific monitoring and therapeutics.