Immobilized pH Gradient Strips Research Articles

Two-Dimensional Gel Electrophoresis in Studies of Flower and Leaf Proteome of Common Buckwheat.

Two-dimensional gel electrophoresis (2-DE) is a proteomic tool used for the separation of protein mixtures according to protein isoelectric point and molecular mass. Although gel-free quantitative and qualitative proteomic study techniques are now available, 2-DE remains a useful analytical tool. The presented protocol was performed to analyze the flower and leaf proteome of common buckwheat using 24cm immobilized pH gradient strips (pH4-7) and visualization of proteins on gels via colloidal Coomassie G-250 staining.

Quantitative assessment confirms deep proteome analysis by integrative top-down proteomics.

The goal of integrative top-down proteomics (i.e., two-dimensional gel electrophoresis [2DE] coupled with liquid chromatography and tandem mass spectrometry [LC/MS/MS]) is a routine analytical approach that fully addresses the breadth and depth of proteomes. To accomplish this, there should be no addition, removal, or modification to any constituent proteoforms. To address two-decade old claims of protein losses during front-end proteome resolution using 2DE, here we tested an alternate rehydration method for immobilized pH gradient strips prior to isoelectric focusing (IEF; i.e., faceup compared to facedown) and quantitatively assessed losses during the front-end of 2DE (rehydration and IEF). Using a well-established high-resolution, quantitative 2DE protocol, there were no detectable proteoform losses using the alternate faceup rehydration method. Although there is a <0.25% total loss of proteoforms during standard facedown rehydration, it is insignificant in terms of having any effect on overall proteome resolution (i.e., total spot count and total spot signal). This report is another milestone in integrative top-down proteomics, disproving long-held dogma in the field and confirming that quantitative front-end 2DE/LC/MS/MS is currently the only method to broadly and deeply analyze proteomes by resolving their constituent proteoforms.

Sometimes faster can be better: Microneedling IPG strips enables higher throughput for integrative top-down proteomics.

Passive rehydration of immobilized pH gradient (IPG) strips for two-dimensional gel electrophoresis (2DE) has, to our knowledge, never been quantitatively evaluated to determine an ideal rehydration time. Seeking to increase throughput without sacrificing analytical rigor, we report that a substantially shorter rehydration time is accomplished when surface area of IPG strips is increased via microneedling. Rehydration for 4h, post microneedling, provides comparable results to overnight rehydration in final analyses by 2DE, while also shortening the overall protocol by 1 day.

Spatial and Pregnancy-Related Changes in the Protein, Amino Acid, and Carbohydrate Composition of Bovine Oviduct Fluid.

Knowledge of how the biochemical composition of the bovine oviduct is altered due to the oviduct anatomy or the presence of an embryo is lacking. Thus, the aim of this study was to assess the effect of (І) oviduct anatomy and (ІІ) embryo presence on oviductal fluid (OF) protein, amino acid, and carbohydrate composition. Cross-bred beef heifers (n = 19) were synchronized and those in standing estrus were randomly allocated to a cyclic (non-bred) or pregnant (artificially inseminated) group. All heifers were slaughtered on Day 3 after estrus. The oviducts ipsilateral to the corpus luteum from each animal were isolated, straightened and cut, separating ampulla and isthmus. Each portion was flushed with 500 µl of PBS enabling recovery of the oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (8-cell embryos; pregnant group) were located in the isthmus of the oviduct. Samples of flushing medium from the isthmus and ampulla were used for proteomic (n = 2 per group), amino acid (n = 5), and carbohydrate (n = 5) analysis. For proteomic analysis, total protein from cyclic and pregnant samples were labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3–10, 17 cm, Protean® IEF cell system, Bio Rad). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). Images were obtained with a Typhoon 9410 scanner and analyzed with Progenesis SameSpots software v 4.0. Amino acid content in the OF was determined by high performance liquid chromatography (HPLC). Glucose, lactate, and pyruvate were quantified using microfluorometric enzyme-linked assays. For the proteomic assessment, the results of the image analysis were compared by ANOVA. For both amino acid and carbohydrate analyses, statistical analysis was carried out by 2-way ANOVA with the Holm-Sidak nonparametric post hoc analysis. On Day 3 post-estrus, OF composition varied based on (І) anatomical region, where isthmic metabolites were present in lower (i.e., lactate, glycine, and alanine) or higher (i.e., arginine) concentrations compared to the ampulla; and (ІІ) embryo presence, which was correlated with greater, arginine, phosphoglycerate kinase 1, serum albumin, α-1-antiproteinase and IGL@ protein concentrations. In conclusion, data indicate that the composition of bovine OF is anatomically dynamic and influenced by the presence of an early embryo.

Supranutritional Supplementation of Vitamin E Influences the Abundance of Antioxidant Proteins in Postmortem Longissimus Lumborum from Heifers

ObjectivesVitamin E is a lipid-soluble antioxidant that can inhibit lipid oxidation and improve beef color stability. The effect of vitamin E on fresh beef color, from the standpoint of lipid oxidation-induced myoglobin oxidation, have been extensively studied. However, the influence of vitamin E on sarcoplasmic proteome profile of beef skeletal muscles is yet to be investigated. Therefore, the objective of this study was to examine the effect of dietary vitamin E on sarcoplasmic proteome of postmortem beef longissimus lumborum (LL) muscle.Materials and MethodsCrossbred heifers, managed with a GrowSafe feeding system, were fed ad libitum corn-based diet containing either no supplemental (CONT) or 1000 IU vitamin E/heifer per day (VITE) for 89 d. The animals were harvested, and carcasses were chilled. The LL muscle samples were obtained from the carcasses of nine (n = 9) VITE and nine (n = 9) CONT heifers 24 h postmortem. The muscle samples were individually vacuum-packaged and frozen at –80°C for proteome analysis. Sarcoplasmic proteome was analyzed using two-dimensional electrophoresis, employing immobilized pH gradient strips (pH 3–10; 17 cm) in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The gels were scanned, and the digital gel images were analyzed. The protein spots exhibiting more than 1.5-fold intensity differences (P &lt; 0.10) between VITE and CONT were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsFive differentially abundant spots were identified using mass spectrometry, and all the spots were over-abundant in CONT. The proteins in the differentially abundant spots were antioxidant proteins (thioredoxin-dependent peroxide reductase, peroxiredoxin-6, and serum albumin) and glycolytic enzymes (β-enolase and triosephosphate isomerase). The antioxidant proteins minimize oxidation of lipids and proteins in muscle matrix, whereas the glycolytic enzymes generate NADPH, which helps maintain the antioxidant proteins in their reduced forms.ConclusionThe strong antioxidant protection offered by vitamin E could have possibly led to less expression of antioxidant proteins as well as glycolytic enzymes that generate antioxidant metabolites in the VITE group, whereas the lack of such protection in CONT group may have led to increased expression of these proteins in the skeletal muscles.

Supranutritional Supplementation of Vitamin E Influences the Abundance of Antioxidant Proteins in Postmortem Longissimus Lumborum from Heifers

ObjectivesVitamin E is a lipid-soluble antioxidant that can inhibit lipid oxidation and improve beef color stability. The effect of vitamin E on fresh beef color, from the standpoint of lipid oxidation-induced myoglobin oxidation, have been extensively studied. However, the influence of vitamin E on sarcoplasmic proteome profile of beef skeletal muscles is yet to be investigated. Therefore, the objective of this study was to examine the effect of dietary vitamin E on sarcoplasmic proteome of postmortem beef longissimus lumborum (LL) muscle.Materials and MethodsCrossbred heifers, managed with a GrowSafe feeding system, were fed ad libitum corn-based diet containing either no supplemental (CONT) or 1000 IU vitamin E/heifer per day (VITE) for 89 d. The animals were harvested, and carcasses were chilled. The LL muscle samples were obtained from the carcasses of nine (n = 9) VITE and nine (n = 9) CONT heifers 24 h postmortem. The muscle samples were individually vacuum-packaged and frozen at –80°C for proteome analysis. Sarcoplasmic proteome was analyzed using two-dimensional electrophoresis, employing immobilized pH gradient strips (pH 3–10; 17 cm) in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The gels were scanned, and the digital gel images were analyzed. The protein spots exhibiting more than 1.5-fold intensity differences (P < 0.10) between VITE and CONT were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsFive differentially abundant spots were identified using mass spectrometry, and all the spots were over-abundant in CONT. The proteins in the differentially abundant spots were antioxidant proteins (thioredoxin-dependent peroxide reductase, peroxiredoxin-6, and serum albumin) and glycolytic enzymes (β-enolase and triosephosphate isomerase). The antioxidant proteins minimize oxidation of lipids and proteins in muscle matrix, whereas the glycolytic enzymes generate NADPH, which helps maintain the antioxidant proteins in their reduced forms.ConclusionThe strong antioxidant protection offered by vitamin E could have possibly led to less expression of antioxidant proteins as well as glycolytic enzymes that generate antioxidant metabolites in the VITE group, whereas the lack of such protection in CONT group may have led to increased expression of these proteins in the skeletal muscles.

157 Dietary L-arginine supplementation affects boar seminal plasma proteome

There are numerous benefit claims for the supplementation of l-arginine in animal production systems. Its positive effect on fertility has been widely characterised in females of various species; however, the effects on the male reproductive system remain debatable with still unfolding molecular mechanisms. Here we investigated the effects of a dietary l-arginine supplementation on boar semen production outcomes, sperm characteristics, and seminal plasma proteome. Nine mature (20 months of age) Nebraska Index Line boars were individually housed and randomly assigned to a soybean meal-based diet containing 0.77% standard l-arginine (control, n=4) or 1.77% high l-arginine (n=5). Boar semen was collected weekly during the 6-week dietary arginine trial. Semen production outcomes (volume and total spermatozoa) and sperm motion (total and progressive motility) and morphology characteristics were subsequently analysed using a computer-assisted sperm analyzer (CEROS II). On Week 6, the seminal plasma of each boar was obtained by centrifugation at 4°C for proteome analysis. The clarified seminal plasma was precipitated; the purified proteins were resuspended in an appropriate buffer and loaded (300μg) onto an immobilized pH gradient strip (isoelectric point 3-10) for the first-dimension electrophoresis. The second-dimension was subsequently performed (4-20% gradient gel), and all gels were stained with Coomassie R250 dye. The PDQuest software (Bio-Rad, Hercules, CA, USA) was used to detect all significantly differentially expressed protein spots (Student’s t-test with P&amp;lt;0.05). Two-way ANOVA repeated measurements (SPSS Statistics, Chicago, IL, USA) was used to analyse dependent (semen outputs and sperm characteristics) and independent (week) variables. A difference was significant at P&amp;lt;0.05. During the l-arginine-consumption trial, semen volumes and total spermatozoa were not significantly affected (P&amp;gt;0.05). Similarly, various sperm motility (total and progressive motility) and velocity characteristics were not affected (P&amp;gt;0.05). By the end of the feed trial, the average (mean±standard error of the mean) semen volume (389±40mL), the total spermatozoa per ejaculate (39±8×109), the total (82±2%) and progressive (62±3%) sperm motility, the sperm velocity (e.g. average path: 98±7μm s−1), and sperm morphology (e.g. distal droplet: 5±1%) parameters of the control group were not significantly different from the group supplemented with high l-arginine: 393±35mL, 47±4×109, 82±2%, 61±3%, 92±8μm s−1, and 5±0%, respectively (P&amp;gt;0.05). In contrast, the proteome profiles of seminal plasma harvested on the sixth week of diet revealed various changes, characterised by the significantly increased expression of 8 protein spots in seminal plasma samples derived from arginine-fed boars (P&amp;lt;0.05). These results indicate that dietary supplementation of l-arginine does not affect the semen outputs, neither the sperm motility characteristics nor their morphology. However, the proteome profile of the seminal plasma was changed by the presence of l-arginine. The findings may have significant implications for boar fertility, and the identification of these proteins is ongoing. This work was supported by USDA-Agricultural Research Service Biophotonics Initiative #58-6402-3-018.

Two-Dimensional Gel Electrophoresis with Immobilized pH Gradients.

Isoelectric focusing (IEF) is a technique in protein research that has been used since 1975 to separate proteins based on their isoelectric point. When combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis, this procedure allows for high-resolution separation of cellular proteins for analytical purposes. Laboratories perform IEF by (a) using carrier ampholytes that migrate through a gel to create the pH gradient or (b) using immobilized pH gradients (IPG) that contain ampholytes bound covalently to a gel. Here we describe an IEF system that uses immobilized pH gradient (IPG) strips that undergo the desired current and voltage setting to separate proteins based on its charge in the first dimension followed by SDS PAGE to generate a two-dimensional map of serum proteins.

Comparative proteomics analysis of maize (Zea mays) leaves infected by small brown planthopper (Laodelphax striatellus)

Maize rough dwarf disease (MRDD) is a viral disease caused by brown planthopper infestation, and leads to great yield loss, especially in China. Comparative proteomics was performed using maize inbred line Zheng 58 and LN 287. MRDD pathogen was detected as rice black-streaked dwarf virus (RBSDV) by quantitative real time PCR (qRT-PCR) in Shandong Province, China. The modified trichloroacetic acid (TCA)/acetone method was used for soluble protein extraction from leaves. Two-dimensional electrophoresis (2-DE) analysis was performed on 24-cm long, pH 4–7 linear immobilized pH gradient (IPG) strips, and gels were stained with silver and coomassie brilliant blue. We identified 944 proteins expressed in RBSDV infected maize leaves by proteomics approaches. Among these, 44 protein spots that revealed a 1.5-fold difference in intensity were identified by mass spectrometry between mock-inoculated and RBSDV infected samples. Among these, 17 and 26 spots were up-regulated, and 27 and 18 spots were down-regulated in the virus infected samples of Zheng 58 and LN 287, respectively. Differential protein spots were analyzed by mass spectrometry identification, which could be divided into six categories. Furthermore, the expression of stress-related proteins was detected and confirmed by qRT-PCR. This study lays the foundation for further investigations, enabling the enhancement of MRDD resistance in maize.

High Sensitivity Top‐down Proteomics: Coomassie for In‐gel Proteoform Detection Rivals MS‐based Peptide Detection

Several thousands of gene and splice variants, as well as post‐translational modifications, deepen the immense complexity of the human proteome. As these protein species or proteoforms define biological mechanisms in health and disease their direct analysis is critical, necessitating top‐down assessments in which the complete amino acid sequence and all modifications remain intact. Two‐dimensional gel electrophoresis (2DE) coupled with downstream mass spectrometry (MS) remains the most thorough and practical approach to Top‐down Discovery Proteomics, with resolving power to routinely analyze complex mixtures of intact proteoforms [1]. Increasing the quality and quantity of 2DE‐derived data largely relies on developing, optimising, and rigorously characterising methods for in‐gel total protein detection, providing quantitative, sensitive detection over a wide dynamic range while remaining practical [2]. Modified colloidal Coomassie Brilliant Blue (cCBB) staining meets these criteria [3, 4]; cCBB provides (sub)femtomole detection sensitivity for SDS‐PAGE and 2DE‐resolved intact proteoforms using either near‐infrared fluorescence detection (nIRFD) or densitometry [5]. This thus rivals the detection sensitivity of peptides by MS in ‘bottom‐up’ (i.e. shotgun) approaches to proteomics, which appear to provide larger protein catalogues but at the expense of critical data concerning proteoforms. Our ongoing work addresses a number of experimental variables which impact detection sensitivity in standard 1 mm‐thick polyacrylamide gels, providing a more realistic baseline measure of cCBB detection sensitivity and enhancing the overall practicality of 2DE for comprehensive proteome analysis. Here we showcase our most recent findings, including preliminary findings of further improvements to cCBB detection sensitivity when using ultra‐thin polyacrylamide gels. The novel ultra‐thin method utilised has been developed from minimally modified commercial equipment for vertical mini‐format gel electrophoresis and is thus accessible, affordable, and simple to implement in laboratories equipped for standard SDSPAGE and 2DE. The ultra‐thin gels do not require backing supports for handling, and potentially are compatible with commercial immobilised pH gradient strips routinely used for first dimension isoelectric focussing, further adding to the value and practicality of the method. Ultra‐thin 2DE may thus enable further mining of 2DE‐resolved proteomes, including the application of high resolution gel‐based proteomics in situations of limited sample quantity (e.g. biopsies, and perhaps single cells).Support or Funding InformationNN and PSA received scholarship support from the WSU Molecular Medicine Research Group and the WSU School of Medicine, respectively. JRC notes the support of an anonymous private family foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Routine 'Top-Down' Approach to Analysis of the Human Serum Proteome.

Serum provides a rich source of potential biomarker proteoforms. One of the major obstacles in analysing serum proteomes is detecting lower abundance proteins owing to the presence of hyper-abundant species (e.g., serum albumin and immunoglobulins). Although depletion methods have been used to address this, these can lead to the concomitant removal of non-targeted protein species, and thus raise issues of specificity, reproducibility, and the capacity for meaningful quantitative analyses. Altering the native stoichiometry of the proteome components may thus yield a more complex series of issues than dealing directly with the inherent complexity of the sample. Hence, here we targeted method refinements so as to ensure optimum resolution of serum proteomes via a top down two-dimensional gel electrophoresis (2DE) approach that enables the routine assessment of proteoforms and is fully compatible with subsequent mass spectrometric analyses. Testing included various fractionation and non-fractionation approaches. The data show that resolving 500 µg protein on 17 cm 3–10 non-linear immobilised pH gradient strips in the first dimension followed by second dimension resolution on 7–20% gradient gels with a combination of lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate (SDS) detergents markedly improves the resolution and detection of proteoforms in serum. In addition, well established third dimension electrophoretic separations in combination with deep imaging further contributed to the best available resolution, detection, and thus quantitative top-down analysis of serum proteomes.

Deep coverage of the beer proteome

We adopted an approach based on peptide immobilized pH gradient-isoelectric focusing (IPG-IEF) separation, coupled with LC-MS/MS, in order to maximize coverage of the beer proteome. A lager beer brewed using traditional Czech technology was degassed, desalted and digested. Tryptic peptides were separated by isoelectric focusing on an immobilized pH gradient strip and, after separation, the gel strip was divided into seven equally sized parts. Peptides extracted from gel fractions were analyzed by LC-MS/MS. This approach resulted in a three-fold increase in the number of proteins identified (over 1700) when compared to analysis of unfractionated beer processed by a filter-aided sample preparation (FASP). Over 1900 protein groups (PGs) in total were identified by both approaches. SignificanceThe study significantly extends knowledge about the beer proteome and demonstrates its complexity. Detailed knowledge of the protein content, especially gluten proteins, will enhance the evaluation of potential health risks related to beer consumption (coeliac disease) and will contribute to improving beer quality.

A semi-virtual two dimensional gel electrophoresis: IF–ESI LC-MS/MS

A method for increasing the productivity of ESI LC-MS/MS (electrospray ionization-liquid chromatography-tandem mass spectrometry) was proposed and applied.After IF (isoelectric focusing) of the sample using IPG (immobilized pH gradient) strip, the strip was cut to sections, and every section was treated according to trypsinolysis protocol for MS/MS analysis. The peptides produced were further analyzed by ESI LC-MS/MS. The procedure allows to:•identify many more proteins and proteoforms compared to shotgun analysis of extracts.•build a semi-virtual 2DE map of identified proteins.

An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins.

The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for different diseases, which may help to identify the proteins that may serve as markers for diagnostics as well as targets for development of new therapeutic potential.

74 THE BOVINE EMBRYO INFLUENCES THE PROTEOME OF THE OVIDUCTAL FLUID

It has been shown that the equine embryo is able to modulate the proteome of the oviduct, increasing the presence of certain proteins involved in the embryo-maternal communication (Smits et al. 2016 Reprod. Fertil. Dev. doi: 10.1071/RD15481). In cattle, the presence of a single embryo did not affect the transcriptome of the oviduct, whereas multiple embryos induced changes (Maillo et al. 2015 Biol. Reprod. 92, 144). The aim of the present study was to examine the effect of the presence of an embryo on the oviduct fluid proteome. Cross-bred beef heifers were synchronized, and those in standing oestrus were randomly allocated to cyclic, not bred (n = 7), or pregnant, artificially inseminated (n = 11), groups. All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut, separating ampulla and isthmus. Each portion was flushed with 500 μL of PBS free of protein to confirm the presence of an oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (pregnant group) were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Flushings from 4 cyclic and 4 pregnant heifers (8-cell embryos) were used for proteomic analysis, including blank controls of PBS. Comparison of ipsilateral ampulla and isthmus in pregnant and cyclic heifers did not reveal any differences. Therefore, samples from ipsilateral oviducts were compared between cyclic and pregnant heifers. Total protein (150 μg) from cyclic and pregnant samples was labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3–10, 17 cm, Protean® IEF cell system, Bio Rad, Hercules, CA, USA). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). The images were obtained with the Typhoon 9410 scanner and analysed with the Progenesis SameSpots software v. 4.0. The results of image analysis showed 42 different spots between the 2 groups (ANOVA P &lt; 0.01 and fold difference &gt;1.5), the majority (38) of which were more abundant in pregnant heifers. Gels were stained with Coomassie blue to detect the spots. The differential spots were digested with trypsin and analysed with Agilent Ion-Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray interface. Twenty spots were highly abundant in pregnant heifers. From these, 3 different proteins were identified with score &gt;5.0 and % SPI &gt;76.0 by mass spectrometry, corresponding to SERPINA1, serum albumin, and serum transferrin. These proteins are abundant in plasma, suggesting that the embryo may be able to activate pathways to increase the permeability of the vascular endothelium in the oviduct. The same proteins were also identified in the equine oviducal proteome in the presence of an embryo, suggesting a possible conserved mechanism between species. In conclusion, presence of an embryo in the bovine oviduct increases the abundance of some proteins that may be related with its early development. This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL-2012–37510 and AGL-2015–70140-R) and Science Foundation Ireland (13/IA/1983).

Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics

The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein ‘A’ on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging. Virtual 2D gel/MS provides access to these intact masses.Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput MS with integrated ‘top-down’ MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5min. Gel-wide chemical and enzymatic methods with further interrogation by MALDI MS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of ‘top-down’ and ‘bottom-up’ proteomics.

Capillary isoelectric focusing-mass spectrometry: Coupling strategies and applications.

IEF on immobilized pH gradient strips is a widespread tool for protein separation, especially as first dimension in commonly utilized 2DE. In the latter arrangement, separations are based on two orthogonal molecular characteristics according to pI in the first and molecular weight in the second dimension. However, the approach is time consuming, quantification is difficult and MS can be applied only offline. Capillary IEF and related IEF techniques in combination with MS provide similar information. The major benefits are high mass resolution and mass accuracy, reproducibility, speed, automation, and quantification by using a high-resolution mass spectrometer. However, online hyphenation of CIEF with MS is interfered by the ampholytes, acids, and bases needed for high-resolution IEF. This review will give an overview about important coupling techniques, like low ampholyte concentration, interim separation by chromatography, or the use of a dialysis interface to separate the analytes from interfering substances. It is focused on strategies which allow sensitive MS detection of CIEF-separated analytes. In addition, proteomic and biopharmaceutical applications of capillary IEF techniques combined with MS are briefly summarized.

Using 7 cm immobilized pH gradient strips to determine levels of clinically relevant proteins in wheat grain extracts.

The aim of the work was to test a relatively simple proteomics approach based on phenol extraction and two-dimensional gel electrophoresis (2-DE) with 7 cm immobilized pH gradient strips for the determination of clinically relevant proteins in wheat grain. Using this approach, 157 2-DE spots were quantified in biological triplicate, out of which 55 were identified by matrix-assisted laser desorption/ionization – time of flight tandem mass spectrometry. Clinically relevant proteins associated with celiac disease, wheat dependent exercise induced anaphylaxis, baker’s asthma, and food allergy, were detected in 24 2-DE spots. However, alcohol-soluble gliadins were not detected with this approach. The comparison with a recent quantitative study suggested that gel-based and gel-free proteomics approaches are complementary for the detection and quantification of clinically relevant proteins in wheat grain.

PTH-092 Discovery and quantitation of novel liver fibrosis biomarkers using proteomics

Introduction Liver biopsy is the reference standard for assessing liver fibrosis and serum biomarkers can be used as a less invasive approach. Various antibody-based assays for serum biomarkers are currently in use to help diagnose fibrosis stage. However, these immunoassays have potential disadvantages such as the inability to detect degraded proteins and often these assays are time consuming. We have developed an antibody-free method to detect and quantify novel liver fibrosis biomarkers in human plasma/serum which overcomes these disadvantages. Method Novel liver fibrosis biomarkers were identified by analysing proteins in plasma/serum samples from controls and patients with varying stages of liver fibrosis using a proteomics technique: two dimensional gel electrophoresis (2DE). For the most promising liver fibrosis biomarkers, an antibody-free assay (parallel reaction monitoring using mass spectrometry) was used which detects tryptic peptides of the biomarkers and their fragments. A calibration curve, established from known amounts of synthetic isotopically labelled peptides, was used to determine the concentrations of our biomarkers in serum/plasma samples from patients with varying stages of liver fibrosis. Results Several candidate biomarkers for hepatic fibrosis were identified using 2DE. Our best biomarkers were promising when compared by Western blotting to the proteins used in existing serum biomarker tests for liver fibrosis. Using parallel reaction monitoring, antibody-free assays were developed for our most promising biomarkers which were able to successfully discriminate between neighbouring stages of liver fibrosis. Conclusion We have developed a fast, sensitive and robust antibody-free method to detect and quantify novel liver fibrosis biomarkers in human plasma/serum. Unlike immunoassays which are restricted on the number of biomarkers due to antibody cost, our method can successfully detect and quantify more than 50 biomarkers in a single 30 min run. This novel assay may help clinicians to assess hepatic fibrosis and reduce the need for invasive liver biopsies. Disclosure of interest None Declared. References Bevin Gangadharan et al . Discovery of novel biomarker candidates for liver fibrosis in hepatitis C patients. PLoS One 2012;7:e39603 Bevin Gangadharan et al . Two dimensional gel electrophoresis using pH3-5.6 immobilised pH gradient strips identifies potential novel disease biomarkers. Nature Protocol Exchange , 2011:doi:10.1038/protex.2011.261 Bevin Gangadharan et al . New approaches for biomarker discovery: The search for liver fibrosis markers in hepatitis C patients. J Proteome Res 2011;10:2643 Bevin Gangadharan et al . Novel serum biomarker candidates for liver fibrosis in hepatitis C patients. Clin Chem 2007;53:1792

Time to dig deep into the plant proteome: a hunt for low-abundance proteins.

Two-dimensional gel electrophoresis (2-DGE) has come a long way since its introduction around 40 years by the pioneering work of these three researchers (Klose, 1975; O'Farrell, 1975; Scheele, 1975). 2-DGE was one of the major breakthroughs in proteomics, enabling researchers to detect, analyze and identify the whole set of proteins of a cell or tissue, simultaneously. With the advancement in technology, some modifications to this technique like development of immobilized pH gradient (IPG) strips were introduced, which undoubtedly, made this technique more simple, rapid and autonomous (Bjellqvist et al., 1982). After its introduction to the present, 2-DGE has been the method of choice for analyzing the complex proteomes of plants. 2-DGE has been used extensively to investigate the effects of biotic and abiotic stress, role of hormones, and developmental changes of plants, among others (Agrawal and Rakwal, 2008). However, it was slowly realized that identification of the plant proteins led to the repeated detection of high-abundance proteins (HAPs) including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and other housekeeping proteins, which are present at 106-105 order of magnitude (Gygi et al., 2000; Patterson and Aebersold, 2003; Gorg et al., 2004). Signaling and other regulatory proteins are generally present 100 molecules per cells. Subsequently, these proteins are difficult to identify by either gel-based or gel-free proteomic approaches, even with access to the latest mass spectrometers. RuBisCO comprises of a large percentage in the total proteins and thus hinders the absorption of low-abundance proteins (LAPs) on the IPG strips, which subsequently results in the poor detection and identification of LAPs on 2D gels and by mass spectrometry (MS), respectively. Therefore, the time has come for all plant proteomers to realize the need to hunt for the LAPs, moving one step ahead from the present. As RuBisCO is the major HAP in plant leaves, here we recommend the incorporation of a RuBisCO depletion/removal method in every plant protein extraction step to look deeper into the plant proteome. RuBisCO depletion will definitely improve the proteome coverage and will lead to the detection of novel unidentified LAPs.