Prolonged chewing of betel quid is known to cause oral diseases, including cancer. The present study was performed to screen for aberrant proteins in the saliva of habitual betel quid chewers compared to nonchewers. Saliva of female subjects (n = 10) who had been chewing betel quid for more than 20 years and nonbetel quid chewers (n = 10) of the same gender and range of age was analyzed by gel-based proteomics. Increased structural microheterogeneity of saliva haptoglobin beta chains indicated by shifts of focused spots similar to that earlier reported in patients with oral squamous cell carcinoma, and their relatively higher abundance compared to nonbetel quid chewers, were detected in saliva protein profiles of all chewers. In addition, the majority of the betel quid chewers also showed significant higher abundance of hemopexin, alpha-1B glycoprotein, alpha1-antitrypsin, complement C3, and transthyretin. These proteins had previously been associated with several different cancers. Our data demonstrated different forms of protein aberration in the saliva of betel quid chewers, which may be indicative of early oral precancerous conditions.
Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and accumulate in shellfish including mussels, oysters, and scallops, causing possible fatalities when inadvertently consumed. Monitoring of PST content of shellfish is therefore important for food safety, with currently approved methods based on HPLC, using pre- or postcolumn oxidation for fluorescence detection (HPLC-FLD). CE is an attractive alternative for screening and detection of PSTs as it is compatible with miniaturization and could be implemented in portable instrumentation for on-site monitoring. In this study, CE methods were developed for C(4) D, FLD, UV absorption detection, and MS-making this first report of C(4) D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used in combination with FLD. The developed CZE-UV and CZE-C(4) D methods provide better resolution, selectivity, and separation efficiency compared to CZE-MS and MEKC-FLD. The sensitivity of the CZE-C(4) D and MEKC-FLD methods was superior to UV and MS, with LOD values ranging from 140 to 715 ng/mL for CZE-C(4) D and 60.9 to 104 ng/mL for MEKC-FLD. With the regulatory limit for shellfish samples of 800 ng/mL, the CZE-C(4) D and MEKC-FLD methods were evaluated for the screening and detection of PSTs in shellfish samples. While the CZE-C(4) D method suffered from significant interferences from the shellfish matrix, MEKC-FLD was successfully used for PST screening of a periodate-oxidized mussel sample, with results confirmed by HPLC-FLD. This confirms the potential of MEKC-FLD for screening of PSTs in shellfish samples.
Alternating-current electro-osmosis, a phenomenon of fluid transport due to the interaction between an electrical double layer and a tangential electric field, has been used both for inducing fluid movement and for the concentration of particles suspended in the fluid. This offers many advantages over other phenomena used to trap particles, such as placing particles at an electrode centre rather than an edge; benefits of scale, where electrodes hundreds of micrometers across can trap particles from the molecules to cells at the same rate; and a trapping volume limited by the vortex height, a phenomenon thus far unstudied. In this paper, the collection of particles due to alternating-current electro-osmosis driven collection is examined for a range of particle concentrations, inter-electrode gap widths, chamber heights and media viscosity and density. A model of collection behaviour is described where particle collection over time is governed by two processes, one driven by the vortices and the other by sedimentation, allowing the determination of the maximum height of vortex-driven collection, but also indicates how trapping is limited by high particle concentrations and fluid velocities. The results also indicate that viscosity, rather than density, is a significant governing factor in determining the trapping behaviour of particles.
This study aims to identify cancer-associated proteins in the secretome of oral cancer cell lines. We have successfully established four primary cell cultures of normal cells with a limited lifespan without human telomerase reverse transcriptase (hTERT) immortalization. The secretome of these primary cell cultures were compared with that of oral cancer cell lines using 2DE. Thirty five protein spots were found to have changed in abundance. Unambiguous identification of these proteins was achieved by MALDI TOF/TOF. In silico analysis predicted that 24 of these proteins were secreted via classical or nonclassical mechanisms. The mRNA expression of six genes was found to correlate with the corresponding protein abundance. Ingenuity Pathway Analysis (IPA) core analysis revealed that the identified proteins were relevant in, and related to, cancer development with likely involvements in tumor growth, metastasis, hyperproliferation, tumorigenesis, neoplasia, hyperplasia, and cell transformation. In conclusion, we have demonstrated that a comparative study of the secretome of cancer versus normal cell lines can be used to identify cancer-associated proteins.
Replicative senescence and stress-induced premature senescence (SIPS) cells are known to share certain traits. However, whether these cells are different at the protein level is unclear. Thus, this study has utilized proteomics to identify differences in the proteomes of replicative senescence and SIPS cells compared to normal cells. Replicative senescence was induced by serial passage of normal cells in culture. SIPS was established by exposure to H2 O2 at a subcytotoxic concentration of 20 μM for two weeks. Following 2DE, protein profiles were compared and protein spots that changed in abundance were identified by MALDI-TOF MS. Quantitative real-time RT-PCR was then performed to evaluate the transcript expression of selected altered proteins. A total of 24 and 10 proteins were found to have changed in abundance in replicative senescence and SIPS cells, respectively, when compared to young cells. Quantitative RT-PCR revealed that nine genes showed the same direction of change as observed in the proteomics analysis. Very little overlap was observed between proteins that changed in replicative senescence and SIPS cells, suggesting that although both SIPS and replicative senescence cells share hallmarks of cellular senescence, they were different in terms of proteins that changed in abundance.
Confirmation of oral squamous cell cancer (OSCC) currently relies on histological analysis, which does not provide clear indication of cancer development from precancerous lesions. In the present study, whole saliva proteins of patients with OSCC (n = 12) and healthy subjects (n = 12) were separated by 2DE to identify potential candidate biomarkers that are much needed to improve detection of the cancer. The OSCC patients' 2DE saliva protein profiles appeared unique and different from those obtained from the healthy subjects. The patients' saliva α1-antitrypsin (AAT) and haptoglobin (HAP) β chains were resolved into polypeptide spots with increased microheterogeneity, although these were not apparent in their sera. Their 2DE protein profiles also showed presence of hemopexin and α-1B glycoprotein, which were not detected in the profiles of the control saliva. When subjected to densitometry analysis, significant altered levels of AAT, complement C3, transferrin, transthyretin, and β chains of fibrinogen and HAP were detected. The increased levels of saliva AAT, HAP, complement C3, hemopexin, and transthyretin in the OSCC patients were validated by ELISA. The strong association of AAT and HAP with OSCC was further supported by immunohistochemical staining of cancer tissues. The differently expressed saliva proteins may be useful complementary biomarkers for the early detection and/or monitoring of OSCC, although this requires validation in clinically representative populations.
Diagnosis of prostate cancer (PCa) is currently much reliant on the invasive and time-consuming transrectal ultrasound-guided biopsy of the prostate gland, particularly in light of the inefficient use of prostate-specific antigen as its biomarker. In the present study, we have profiled the sera of patients with PCa and benign prostatic hyperplasia (BPH) using the gel- and lectin-based proteomics methods and demonstrated the significant differential expression of apolipoprotein AII, complement C3 beta chain fragment, inter-alpha-trypsin inhibitor heavy chain 4 fragment, transthyretin, alpha-1-antitrypsin, and high molecular weight kininogen (light chain) between the two groups of patients' samples. Our data are suggestive of the potential use of the serum proteins as complementary biomarkers to effectively discriminate PCa from BPH, although this requires further extensive validation on clinically representative populations.
We barcoded 25 in vitro isolates (representing 92 samples) of Giardia duodenalis from humans and other animals, which have been assembled by the Upcroft team at the Queensland Institute of Medical Research over a period of almost three decades. We used mutation scanning-coupled sequencing of loci in the triosephosphate isomerase, glutamate dehydrogenase and β-giardin genes, combined with phylogenetic analysis, to genetically characterise them. Specifically, the isolates (n514) of G. duodenalis from humans from Australia (AD113; BRIS/83/HEPU/106; BRIS/87/HEPU/713; BRIS/89/HEPU/1003; BRIS/92/HEPU/1541; BRIS/92/HEPU/1590; BRIS/92/HEPU/2443; BRIS/93/HEPU/1706), Malaysia (KL/92/IMR/1106) and Afghanistan (WB), a cat from Australia (BAC2), a sheep from Canada (OAS1) and a sulphur-crested cockatoo from Australia (BRIS/95/HEPU/2041) represented assemblage A (sub-assemblage AI-1, AI-2 or AII-2); isolates (n510) from humans from Australia (BRIS/91/HEPU/1279; BRIS/92/HEPU/2342; BRIS/92/HEPU/2348; BRIS/93/HEPU/1638; BRIS/93/HEPU/1653; BRIS/93/HEPU/1705; BRIS/93/HEPU/1718; BRIS/93/HEPU/1727), Papua New Guinea (BRIS/92/HEPU/1487) and Canada (H7) represented assemblage B (sub-assemblage BIV) and an isolate from cattle from Australia (BRIS/92/HEPU/1709) had a match to assemblage E. Isolate BRIS/90/HEPU/1229 from a human from Australia was shown to represent a mixed population of assemblages A and B. These barcoded isolates (including stocks and derived lines) now allow direct comparisons of experimental data among laboratories and represent a massive resource for transcriptomic, proteomic, metabolic and functional genomic studies using advanced molecular technologies.
Bacterial secreted proteins are known to be involved in virulence and may mediate important host-pathogen interactions. In this study, when the stationary phase culture supernatant of Burkholderia pseudomallei was subjected to 2-DE, 113 protein spots were detected. Fifty-four of the secreted proteins, which included metabolic enzymes, transcription/translation regulators, potential virulence factors, chaperones, transport regulators, and hypothetical proteins, were identified using MS and database search. Twelve of these proteins were apparently reactive to antisera of mice that were immunised with B. pseudomallei secreted proteins. These proteins might be excellent candidates to be used as diagnostic markers or putative candidate vaccines against B. pseudomallei infections.
Toxoplasma gondii infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. To identify the potential infection markers of this disease, 2-DE and Western blot methods were employed to study the parasite circulating antigens and host-specific proteins in the sera of T. gondii-infected individuals. The comparisons were made between serum protein profiles of infected (n=31) and normal (n=10) subjects. Antigenic proteins were identified by immunoblotting using pooled sera and monoclonal anti-human IgM-HRP. Selected protein spots were characterised using mass spectrometry. Prominent differences were observed when serum samples of T. gondii-infected individuals and normal controls were compared. A significant up-regulation of host-specific proteins, α(2)-HS glycoprotein and α(1)-B glycoprotein, was also observed in the silver-stained gels of both active and chronic infections. However, only α(2)-HS glycoprotein and α(1)-B glycoprotein in the active infection showed immunoreactivity in Western blots. In addition, three spots of T. gondii proteins were detected, namely (i) hypothetical protein chrXII: 3984434-3 TGME 49, (ii) dual specificity protein phosphatase, catalytic domain TGME 49 and (iii) NADPH-cytochrome p450 reductase TGME 49. Thus, 2-DE approach followed by Western blotting has enabled the identification of five potential infection markers for the diagnosis of toxoplasmosis: three are parasite-specific proteins and two are host-specific proteins.
Binding constants for the enantiomers of modafinil with the negatively charged chiral selector sulfated-β-CD (S-β-CD) using CE technique is presented. The calculations of the binding constants employing three different linearization plots (double reciprocal, X-reciprocal and Y-reciprocal) were performed from the electrophoretic mobility values of modafinil enantiomers at different concentrations of S-β-CD in the BGE. The highest inclusion affinity of the modafinil enantiomers were observed for the S-enantiomer-S-β-CD complex, in agreement with the computational calculations performed previously. Binding constants for each enantiomer-S-β-CD complex at different temperatures, as well as thermodynamic parameters for binding, were calculated. Host-guest binding constants using the double reciprocal fit showed better linearity (r(2)>0.99) at all temperatures studied (15-30°C) and compared with the other two fit methods. The linear van't Hoff (15-30°C) plot obtained indicated that the thermodynamic parameters of complexation were temperature dependent for the enantiomers.
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.
Rapid and direct online preconcentration followed by CE with capacitively coupled contactless conductivity detection (CE-C(4)D) is evaluated as a new approach for the determination of glyphosate, glufosinate (GLUF), and aminophosphonic acid (AMPA) in drinking water. Two online preconcentration techniques, namely large volume sample stacking without polarity switching and field-enhanced sample injection, coupled with CE-C(4)D were successfully developed and optimized. Under optimized conditions, LODs in the range of 0.01-0.1 microM (1.7-11.1 microg/L) and sensitivity enhancements of 48- to 53-fold were achieved with the large volume sample stacking-CE-C(4)D method. By performing the field-enhanced sample injection-CE-C(4)D procedure, excellent LODs down to 0.0005-0.02 microM (0.1-2.2 microg/L) as well as sensitivity enhancements of up to 245- to 1002-fold were obtained. Both techniques showed satisfactory reproducibility with RSDs of peak height of better than 10%. The newly established approaches were successfully applied to the analysis of glyphosate, glufosinate, and aminophosphonic acid in spiked tap drinking water.
An efficient method for the simultaneous enantioseparation of cyproconazole, bromuconazole, and diniconazole enantiomers was developed by CD-modified MEKC using a dual mixture of neutral CDs as chiral selector. Three neutral CDs namely hydroxypropyl-beta-CD, hydroxypropyl-gamma-CD, and gamma-CD were tested as chiral selectors at different concentrations ranging from 10, 20, 30 and 40 mM, but enantiomers of the studied fungicides were not completely separated. The best dual chiral recognition mode for the simultaneous separation of cyproconazole, bromuconazole, and diniconazole enantiomers was achieved with a mixture of 27 mM hydroxypropyl-beta-CD and 3 mM hydroxypropyl-gamma-CD in 25 mM phosphate buffer (pH 3.0) containing 40 mM SDS to which methanol-acetonitrile (10%:5% v/v) was added as organic modifiers. The best separation was based on the appearance of 10 peaks simultaneously, with good resolution (R(s) 1.1-15.9), and peak efficiency (N>200,000). Good repeatabilities in the migration time, peak area, and peak height were obtained in terms of RSD ranging from (0.72 to 1.06)%, (0.39 to 3.49)%, and (1.90 to 4.84)%, respectively.
A 35 kDa glycoprotein whose abundance was previously demonstrated to be enhanced in sera of patients with endometrial adenocarcinoma (n = 12), was isolated from pooled sera of three of the cancer patients using champedak galactose-binding lectin affinity chromatography in the present study. Subjecting it to 2-DE and MS/MS, the glycoprotein was identified as the O-glycosylated fragment of inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4). When compared to control sera (n = 17), expression of the 35 kDa ITIH4 cleavage fragment was demonstrated to be significantly enhanced in sera of patients with breast carcinoma (n = 10), epithelial ovarian carcinoma (n = 10), and germ cell ovarian carcinoma (n = 10) but not in patients with nasopharyngeal carcinoma (n = 13) and osteosarcoma (n = 7). The lectin-based electrophoretic bioanalytical method adopted in the present study may be used to assess the physiological relevance of ITIH4 fragmentation and its correlation with different malignancies, their stages and progression.
The expression of high-abundance serum proteins in newly diagnosed patients with endometrial adenocarcinoma (EACa), squamous cell cervical carcinoma (SCCa) and cervical adenocarcinoma (ACCa), relative to control female subjects, was analyzed by subjecting serum samples to 2-DE followed by image analysis of the silver-stained protein profiles. The three cohorts of cancer patients demonstrated different altered expression of serum high-abundance proteins compared to negative control women. The expression of alpha1-antitrypsin, alpha1-B glycoprotein, cleaved high-molecular-weight kininogen (light chain) and antithrombin III were consistently altered in all the patients. However, clusterin was upregulated only in the patients with EACa, while those with SCCa and ACCa were typically characterized by the upregulated expression of zinc alpha-2-glycoprotein. The aberrant expression of selective serum proteins in the various cohorts of cancer patients was validated by competitive ELISA as well as by lectin detection. Analysis by using the champedak galactose binding lectin further highlighted an unidentified protein that may be differently glycosylated in the sera of the EACa patients that were studied.
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.
Hepatitis B core antigen (HBcAg) expressed in Escherichia coli is able to self-assemble into large and small capsids comprising 240 (triangulation number T = 4) and 180 (triangulation number T = 3) subunits, respectively. Conventionally, sucrose density gradient ultracentrifugation and SEC have been used to separate these capsids. However, good separation of the large and small particles with these methods is never achieved. In the present study, we employed a simple, fast, and cost-effective method to separate the T = 3 and T = 4 HBcAg capsids by using native agarose gel electrophoresis followed by an electroelution method (NAGE-EE). This is a direct, fast, and economic method for isolating the large and small HBcAg particles homogenously based on the hydrodynamic radius of the spherical particles. Dynamic light scattering analysis demonstrated that the T = 3 and T = 4 HBcAg capsids prepared using the NAGE-EE method are monodisperse with polydispersity values of ∼15% and ∼13%, respectively. ELISA proved that the antigenicity of the capsids was not affected in the purification process. Overall, NAGE-EE produced T = 3 and T = 4 capsids with a purity above 90%, and the recovery was 34% and 50%, respectively (total recovery of HBcAg is ∼84%), and the operation time is 15 and 4 times lesser than that of the sucrose density gradient ultracentrifugation and SEC, respectively.
Matched MeSH terms: Electrophoresis, Agar Gel/methods*
The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required.
Dielectrophoresis is the movement of particles in nonuniform electric fields and has been of interest for application to manipulation and separation at and below the microscale. This technique has the advantages of being noninvasive, nondestructive, and noncontact, with the movement of particle achieved by means of electric fields generated by miniaturized electrodes and microfluidic systems. Although the majority of applications have been above the microscale, there is increasing interest in application to colloidal particles around a micron and smaller. This paper begins with a review of colloidal and nanoscale dielectrophoresis with specific attention paid to separation applications. An innovative design of integrated microelectrode array and its application to flow-through, continuous separation of colloidal particles is then presented. The details of the angled chevron microelectrode array and the test microfluidic system are then discussed. The variation in device operation with applied signal voltage is presented and discussed in terms of separation efficiency, demonstrating 99.9% separation of a mixture of colloidal latex spheres.