Solution-phase Isoelectric Focusing Research Articles

A proteomic analysis of Curcuma comosa Roxb. rhizomes.

BackgroundThe similarly in plant physiology and the difficulty of plant classification, in some medicinal plant species, especially plants of the Zingiberaceae family, are a major problem for pharmacologists, leading to mistaken use. To overcome this problem, the proteomic base method was used to study protein profiles of the plant model, Curcuma comosa Roxb., which is a member of the Zingiberaceae and has been used in traditional Thai medicine as an anti-inflammatory agent for the treatment of postpartum uterine bleeding.ResultsDue to the complexity of protein extraction from this plant, microscale solution-phase isoelectric focusing (MicroSol-IEF) was used to enrich and improve the separation of Curcuma comosa rhizomes phenol-soluble proteins, prior to resolving and analyzing by two-dimensional polyacrylamide gel electrophoresis and identification by tandem mass spectrometry. The protein patterns showed a high abundance of protein spots in the acidic range, including three lectin proteins. The metabolic and defense enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase, that are associated with antioxidant activity, were mainly found in the basic region. Furthermore, cysteine protease was found in this plant, as had been previously reported in other Zingiberaceae plants.ConclusionThis report presents the protein profiles of the ginger plant, Curcuma comosa. Several interesting proteins were identified in this plant that may be used as a protein marker and aid in identifying plants of the Zingiberaceae family.

Proteomic profiling combining solution-phase isoelectric fractionation with two-dimensional gel electrophoresis using narrow-pH-range immobilized pH gradient gels with slightly overlapping pH ranges

This paper describes a simple new approach toward improving resolution of two-dimensional (2-D) protein gels used to explore the mammalian proteome. The method employs sample prefractionation using solution-phase isoelectric focusing (IEF) to split the mammalian proteome into well-resolved pools. As crude samples are thus prefractionated by pI range, very-narrow-pH-range 2-D gels can be subsequently employed for protein separation. Using custom pH partition membranes and commercially available immobilized pH gradient (IPG) strips, we maximized the total separation distance and throughput of seven samples obtained by prefractionation. Both protein loading capacity and separation quality were higher than the values obtained by separation of fractionated samples on narrow-pH-range 2-D gels; the total effective IEF separation distance was ~82 cm over the pH range pH 3-10. This improved method for analyzing prefractionated samples on narrow-pH-range 2-D gels allows high protein resolution without the use of large gels, resulting in decreased costs and run times.

Proteomic Alteration During Dormant Period of Curcuma Longa Rhizomes

Proteins involved during storage of Curcuma longa have not been investigated in detail. Proteins and nucleic acids are considered to be essential for the sprouting period. As numerous applications of proteomic approaches have been reported in many areas of biology, biochemistry and biomedicine, we chose to use proteomic technology to study the protein expression of Curcuma longa (Khaminchun) rhizome during dormancy and sprouting. Microscale solution-phase isoelectric focusing (Zoom) was employed to enrich the low abundance proteins in the pH range of 5.4-10 and improve the separation of those proteins in the acidic range from 3-5.4. Samples were drawn at seven-day intervals from harvest until the commencement of sprouting. The proteomic patterns of the storage period (0, 14, 21, 42, and 70 days) were studied and identified by LC/MS/MS in these two pH ranges. High levels of glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme, were present and in glycosylated and phosphorylated forms. Sporamin, the major storage protein of the tuberous roots of sweet potato, was highly expressed in the dormant period and lower expression was detected in the sprouting period. Moreover, the enzyme for catalyzing the synthesis of catechin, leucoanthocyanidin reductase, was found for the first time in the Curcuma longa rhizome. These results represent the first proteomic patterns during the storage period of Curcuma longa. For the rhizomes of Curcuma longa, visible sprouting was observed within 70 days after harvest.

Proteomic Alteration During Storage of Curcuma Longa Rhizomes

The rhizomes of genus Curcuma are extensively used as spices, food preservatives, coloring materials, cosmetics and medicines. The Curcuma becomes dormant during the dry season. The changes in proteins during storage of Curcuma longa have not been investigated in detail. We employed proteomic technology to study the protein expression from the day of harvest to the commencement of sprouting of Curcuma longa. The two dimensional gel electrophoresis patterns (pH 4-7) of the rhizomes showed a high abundance of proteins with pI in the range of 3-5 and low abundance with pI in the range of 5-7. Microscale solution-phase isoelectric focusing (Zoom) was employed to enrich the low abundance proteins in the pH range of 5.4-10 and improve the separation of those proteins in the acidic range from 3-5.4. The total storage period was 77 days. Samples were drawn at an interval of 7 days from the harvest until sprouting. The proteomic patterns of the storage period (0, 7, 14, 21, 35, 42, 49 and 70 days) were studied in these two pH ranges. In the pH range from 3-5.4, the expression of ribulose 1, 5-bisphosphate dehydrogenase, actin, anionic peroxidase swpa4, maturase and photosystem I assembly protein ycf4 were increased and putative oxygen evolving enhance protein I, hypothetical protein MeviPp13 and MLP_like protein 423 were decreased from 0 to 70 days, while in the pH range from 5.4-10, the expression of eight identified proteins were expressed only at 70 days. Sporamin, the major storage protein of the tuberous roots of sweet potato was highly expressed in the dormancy period and lower expression seen in the sprouting period

Proteomic analysis of age dependent nitration of rat cardiac proteins by solution isoelectric focusing coupled to nanoHPLC tandem mass spectrometry

Protein nitration occurs as a result of oxidative stress induced by reactive oxygen (ROS) and reactive nitrogen species (RNS). Therefore, protein nitration serves as a hallmark for protein oxidation in vivo. We have previously reported on age dependent protein nitration in cardiac tissue of Fisher 344 BN-F1 rats analyzed by two-dimensional gel electrophoresis; however, only one specific nitration site was identified [Kanski, J., Behring, A., Pelling, J., Schöneich, C., 2005a. Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging. Am. J. Physiol. Heart Circ. Physiol. 288, H371–381]. In the present report, we used solution phase isoelectric focusing (IEF) followed by nanoHPLC–ESI-MS/MS that allowed us to obtain good MS/MS data to identify specific sites of protein nitration in cardiac tissue. As expected, more nitrated proteins were detected in cardiac tissue of old rats, including myosin heavy chain, neurofibromin, tropomyosin and nebulin-related anchoring protein. The post-translational modification of these cytoskeletal proteins may provide some rationale for the age-dependent functional decline of the heart.

Solution phase isoelectric fractionation in the multi-compartment electrolyser: A divide and conquer strategy for the analysis of complex proteomes

Sample complexity frequently interferes with the analysis of low-abundance proteins by two-dimensional gel electrophoresis (2DGE). Ideally, high abundance proteins should be removed, allowing low-abundance proteins to be applied at much higher concentrations than is possible with the unfractionated sample. One approach is to partition the sample in a manner that segregates the bulk of extraneous proteins from the protein(s) of interest. Solution phase isoelectric focusing in the multi-compartment electrolyser generates fractions of discrete isoelectric point (pI) intervals allowing isolated narrow segments of a proteome to be analysed individually by 2DGE. It is particularly useful for the isolation of low-abundance proteins of extremely basic or acidic pI.

Proteomic characterisation of cell contact-dependent macrophage activation

The generation of tumour necrosis factor alpha (TNF-α) in the rheumatoid synovium is widely perceived as pivotal to disease pathogenesis in rheumatoid arthritis (RA). While TNF-α production in dispersed synovial cell cultures is T-cell-dependent [1] there is little evidence of antigen-dependent T-cell activation. One explanation for this apparent paradox is the well-documented ability of appropriately activated T cells to drive production of TNF-α from macrophages through a cell contact-dependent, antigen-independent mechanism [2]. Despite some promising leads (e.g. CD69 [3]), the identities of the relevant stimulating T-cell surface ligands, and the macrophage signalling pathways engaged following cell contact remain obscure. Our approach has been to develop new proteomic techniques to address these issues in a systematic and unbiased fashion. We used cell surface biotinylation and solution-phase isoelectric focusing to extract and resolve plasma membrane proteins from murine T-cell hybridomas and primary splenocytes prior to identification using liquid chromatography tandem mass spectrometry (LC MS/MS). Using this method we identified 75 plasma membrane proteins, 13 of which appeared to be regulated following stimulation with phorbol 12-myristate 13-acetate and ionomycin. Six of these putative changes were verified independently by western blotting or by flow cytometry [4]. To develop protocols to examine macrophage signalling pathways we utilised anti-phosphotyrosine antibodies to recover tyrosine phosphoproteins from RAW 264.7 cells activated with the tyrosine phosphatase inhibitor, pervanadate. Recovered proteins were eluted using phenyl phosphate, then resolved and identified by solution-phase isoelectric focusing and LC MS/MS. We identified 113 proteins inducibly associated with anti-phosphotyrosine immunoprecipitates from distinct subcellular compartments (cytosol, nucleus and plasma membrane), many of which were known tyrosine phosphoproteins (e.g. Fyn, Syk, SHIP, SLP76) but also included several hypothetical proteins, hitherto not documented at the protein level. We then used this methodology to identify macrophage signalling proteins phosphorylated following cell contact with T-cell receptor transgenic splenocytes stimulated for 48 hours with specific (influenza virus hemaglutinin), or non-specific (ovalbumin), peptide. Proteins recovered in RAW cell phosphotyrosine immunoprecipitates were analysed by western blotting using antibodies to phosphotyrosine or the signalling molecules phospholipase C gamma, Hck, p38 and ERK. Cell contact with either resting or activated splenocytes induced acute and transient tyrosine phosphorylation of the src kinase Hck and phospholipase C gamma. In contrast, sustained activation of the MAP kinases p38 and ERK was observed only after contact with activated splenocytes. Application of these methods should shed new light on the molecular bases of the contact-dependent activation of macrophages by activated T cells and, thereby, suggest novel targets for therapeutic intervention in RA.

Combining microscale solution-phase isoelectric focusing with Multiplexed Proteomics dye staining to analyze protein post-translational modifications.

Previously, a strategy for rapidly identifying mitochondrial phosphoproteins was presented that involves prefractionating multisubunit complexes by sucrose gradient centrifugation, followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and selective staining of phosphoproteins and total protein with fluorescent dyes [1]. Though suitable for evaluating the mitochondrial proteome, which consists of numerous multisubunit complexes, the strategy is not generally applicable to other complex proteomes. We determined that prefractionating samples by solution-phase isoelectric focusing is an effective alternative to sucrose-gradient fractionation that can be applied equally well to the analysis of mitochondrial and plasma proteins. Fluorescence-based multiplexing dye technologies greatly extend the capacity of SDS-polyacrylamide gel electrophoresis with respect to the investigation of proteome-wide changes in protein expression and post-translational modification, such as phosphorylation and glycosylation [2]. Overall, the prefractionation/Multiplexed Proteomics staining technology permits rapid, higher throughput screening of specimens for the identification of potentially interesting fractions that can subsequently be evaluated more thoroughly by two-dimensional gel electrophoresis.

A proteome strategy for fractionating proteins and peptides using continuous free-flow electrophoresis coupled off-line to reversed-phase high-performance liquid chromatography.

Extensive prefractionation is now considered to be a necessary prerequisite for the comprehensive analysis of complex proteomes where the dynamic range of protein abundances can vary from approximately 10(6) for cells to approximately 10(10) for tissues such as blood. Here, we describe a high-resolution 2D protein separation system that uses a continuous free-flow electrophoresis (FFE) device to fractionate complex protein mixtures by solution-phase isoelectric focusing (IEF) into 96 well-defined pools, each separated by approximately 0.02-0.10 pH unit depending on the gradient created, followed by rapid (approximately 6 min per analysis) reversed-phase high-performance liquid chromatography (RP-HPLC) of each FFE pool. Fractionated proteins are readily visualized in a virtual 2D format using software that plots protein loci, pI in the first dimension and relative hydrophobicity (i.e., RP-HPLC retention time) in the second dimension. By coupling a diode-array detector in line with a multiwavelength fluorescence detector, separated proteins can be monitored in the RP-HPLC eluent by both UV absorbance and intrinsic fluorescence simultaneously from a single experiment. Triplicate analyses of standard proteins using a pH 3-10 gradient conducted over a 3-day period revealed a high system reproducibility with a SD of 0.57 (0.05 pH unit) within the FFE pools and 0.003 (0.18 s) for protein retention times in the second-dimension RP-HPLC step. In addition, we demonstrate that the FFE-IEF/RP-HPLC separation strategy can also be applied to complex mixtures of low molecular weight compounds such as peptides. With the facile ability to measure the pH of the isoelectric focused pools, peptide pI values can be estimated and used to qualify peptide identifications made using either MS/MS sequencing approaches or pI discriminated peptide mass fingerprinting. The calculated peak capacity of this 2D liquid-based FFE-IEF/RP-HPLC system is 6720.

Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry.

Identification of the serum proteome is a daunting analytical task due to the complex nature of the sample which has an extremely large dynamic range of protein components. This report addresses this issue by using centrifugal ultrafiltration to enrich the low-molecular-weight (LMW) serum proteome while decreasing the amount of abundant high-molecular-weight proteins. Reduction of the complex nature of the sample was achieved by fractionation of the LMW serum proteins using solution-phase isoelectric focusing (IEF). Multiple enzyme digestions are performed and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis of the tandem mass spectra resulted in the identification of 262 proteins belonging to LMW serum proteome. Our results demonstrate the effectiveness of this methodology to isolate and identify LMW proteins with improved confidence in the MS data acquired. In addition, our methodology can be combined with other multidimensional chromatography techniques performed on the peptide level to increase the number of identified proteins.

Expression Profiling of Lymphocyte Plasma Membrane Proteins

The physicochemical properties of plasma membrane proteins of mammalian cells render them refractory to systematic analysis by two-dimensional electrophoresis. We have therefore used in vivo cell surface labeling with a water-soluble biotinylation reagent, followed by cell lysis and membrane purification, prior to affinity capture of biotinylated proteins. Purified membrane proteins were then separated by solution-phase isoelectric focusing and SDS-PAGE and identified by high-pressure liquid chromatography electrospray/tandem mass spectrometry. Using this approach, we identified 42 plasma membrane proteins from a murine T cell hybridoma and 46 from unfractionated primary murine splenocytes. These included three unexpected proteins; nicastrin, osteoclast inhibitory lectin, and a transmembrane domain-containing hypothetical protein of 11.4 kDa. Following stimulation of murine splenocytes with phorbol ester and calcium ionophore, we observed differences in expression of CD69, major histocompatibility complex class II molecules, the glucocorticoid-induced TNF receptor family-related gene product, and surface immunoglobulin M and D that were subsequently confirmed by Western blot or flow cytometric analysis. This approach offers a generic and powerful strategy for investigating differential expression of surface proteins in many cell types under varying environmental and pathophysiological conditions.