Mapping of alkaline proteins in bovine skeletal muscle

In agricultural sciences, proteomics has become the new hope for analyzing the meat quality traits that are closely related to the skeletal muscle traits. 2-DE muscle maps of many species have been recently reported and used to find molecular markers of meat quality traits. However, one limitation of 2-DE based analyses is due to the limited alkaline protein separation. Considering this problem, there is a need to use recent advances that have markedly improved the 2-DE based analysis of alkaline proteins. Hence, the present study provides additional information concerning the alkaline proteome of bovine skeletal muscle by using an appropriate protocol to characterize proteins over the entire range of pH 7-11. A total of 32 distinct gene products corresponding to 60 protein spots were identified by PMF and grouped in seven categories according to their main function. This 2-D map will contribute to muscle proteome studies since a significant portion of proteins is in the alkaline pH range.     

Toward a high resolution 2-DE profile of the normal human liver proteome using ultra-zoom gels

The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project (HLPP), the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis (2-DE). We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5–5.5, 5–6, 5.5–6.7, 6–9 and 6–11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3–10 and 4–7 pH gradients respectively, we obtained 5481 protein spots from the multiple (overlapping) narrow pH range ultra-zoom gels in the range of pH 4.5–9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.     

Depletion of the highly abundant protein albumin from human plasma using the Gradiflow

Analysis of complex protein samples by two-dimensional electrophoresis (2-DE) is often more difficult in the presence of a few predominant proteins. In plasma, proteins such as albumin mask proteins of lower abundance, as well as significantly limiting the amount of protein that can be loaded onto the immobilized pH gradient strip. In this paper the Gradiflow, a preparative electrophoresis system, has been used to deplete human plasma of the highly abundant protein albumin under native and denatured conditions. A three step protocol incorporating a charge separation to collect proteins with an isoelectric point greater than albumin and two size separations to isolate proteins larger and smaller than albumin, was used. When the albumin depleted fractions were analysed on pH 3-10 2-DE gels, proteins that were masked by albumin were revealed and proteins not seen in the unfractionated plasma sample were visualised. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analysis confirmed the identification of the protein that lies beneath albumin to be C4B-binding protein alpha chain. The liquid fractions from the Gradiflow separations were also analysed by liquid chromatography-tandem mass spectrometry to confirm the proteins were separated according to their size and charge mobility in an electric field.     

Proteomic profiling of hempseed proteins from Cheungsam

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.     

Proteomic profiling of hempseed proteins from Cheungsam

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.     

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up

With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.     

Reproducibility,sensitivity and compatibility of the ProteoExtract® subcellular fractionation kit with saturation labeling of laser microdissected tissues

Laser microdissection (LMD), a method of isolating specific microscopic regions of interest from a tissue that has been sectioned, is increasingly being applied to study proteomics. LMD generally requires tissues to be fixed and histologically stained, which can interfere with protein recovery and subsequent analysis. We evaluated the compatibility and reproducibility of protein extractions from laser microdissected human colon mucosa using a subcellular fractionation kit (ProteoExtract^®, Calbiochem). Four protein fractions corresponding to cytosol (fraction 1), membrane/organelle (fraction 2), nucleus (fraction 3) and cytoskeleton (fraction 4) were extracted, saturation labeled with Cy5 and 5 μg separated by both acidic (pH 4–7) and basic (pH 6–11) 2‐DE. The histological stains and fixation required for LMD did not interfere with the accurate subcellular fractionation of proteins into their predicted fraction. The combination of subcellular fractionation and saturation CyDye labeling produced very well resolved, distinct protein spot maps by 2‐DE for each of the subcellular fractions, and the total number of protein spots consistently resolved between three independent extractions for each fraction was 893, 1128, 1245 and 1577 for fractions 1, 2, 3 and 4, respectively. Although significant carryover of protein did occur between fractions, this carryover was consistent between experiments, and very low inter‐experimental variation was observed. In summary, subcellular fractionation kits are very compatible with saturation labeling DIGE of LMD tissues and provide greater coverage of proteins from very small amounts of microdissected material.     

Profiling of mitochondrial associated proteins from rat colon

Mitochondrial dysfunction, damage and mutations of mitochondrial proteins give rise to a range of ill understood patterns of disease. Although there is significant general knowledge of the proteins and the functional processes of the mitochondria, there is little knowledge of difference about how mitochondria respond and how they are regulated in different organs and tissues. Proteomic profiling of mitochondria and associated proteins involved in mitochondrial regulation and trafficking within cells and tissues has the potential to provide insights into mitochondrial dysfunction associated with many human diseases. The rat colon mitoproteome analysis presented here provides a useful tool to assist in identification and interpretation of mitochondrial dysfunction implicated in colon pathogenesis. 2DPAGE followed by LC/MS/MS was used to identify 430 proteins from mitochondrial enriched fractions prepared from rat colon, resulting in 195 different proteins or approximately 50% of the resolved proteins being identified as multiple protein expression forms. Proteins associated with the colon mitoproteome were involved in calcium binding, cell cycle, energy metabolism and electron transport chain, protein folding, protein synthesis and degradation, redox regulation, structural proteins, signalling and transporter and channel proteins. The mitochondrial associated proteins identified in this study of colon tissue complement and are compared with other recently published mitoproteome analyses from other organ tissues, and will assist in revealing potentially organ specific roles of the mitochondria and organ specific disease associated with mitochondrial dysfunction.     

Characterisation of rice anther proteins expressed at the young microspore stage

In combination with two-dimensional polyacrylamide gel electrophoresis (2-DE) protein mapping and mass spectrometry analysis, the pattern of gene expression in specific tissues at a specific stage can be displayed and characterised. We used this approach for rice (Oryza sativa L. cultivar Doongara) to display and assign identity to proteins in the anthers at the young microspore stage. Over 4000 anther proteins in the pI range of 4-11 and molecular mass range of 6-122 kDa were reproducibly resolved after silver staining, representing about 10% of the estimated total genomic output of rice. Two hundred and seventy-three protein spots have been extracted either from polyninylidene diffluoride membrane blots or from colloidal Coomassie blue stained 2-DE gels and analysed by N-terminal sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS) analysis or tandem MS sequencing. This enabled identification of 53 anther protein spots representing 43 different proteins. Using the publicly available rice expressed sequence tag (EST) database at the National Centre for Biotechnology Information, a further 37 protein spots were matched to ESTs. After BLAST searching with these ESTs, we were able to predict the identity of 22 of these protein spots. Proteome reference maps of rice anthers have been constructed according to the SWISS-2DPAGE standards and are available for public access at http://semele.anu.edu.au/2d/2d.html.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.     

Proteomic analysis of somatic embryogenesis in <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Two dimensional gel electrophoresis coupled to mass spectrometry has been used to study the somatic embryogenesis in Vitis vinifera, by comparing embryogenic and non embryogenic calluses of the Thompson seedless cv. More than 1,000 spots were reproducibly resolved in colloidal Coomassie brilliant blue stained gels over a pI nonlinear range of 3–10 in the first dimension and using homogeneous 12.5% polyacrylamide gels in the second dimension. The expression pattern of 35 spots differed significantly between the two samples. These spots were processed by mass spectrometry analysis and the protein identity was assigned by using both the non-redundant protein and EST databases. Several responsive proteins, some already known to be involved in the somatic embryogenesis process while others, for the first time put into relation with this process, have been described. Moreover, they have been subdivided in functional categories, and their putative role is discussed in terms of their relevance in the somatic embryogenesis process.     

Proteomics strategy based on liquid-phase IEF and 2-D DIGE: application to bone marrow mesenchymal progenitor cells

Global comparative proteomics is a promising new approach with broad application in basic and clinical biological science. Recent advances include the development of 2-D DIGE, a proteomic equivalent to mRNA differential display, in which differentially labeled samples are multiplexed and analyzed by high-resolution 2-DE. This study presents a new 2-D DIGE protocol, in which complex protein samples from normal and leukemic human bone marrow mesenchymal progenitor cells were used as model samples for a novel combination of liquid-phase IEF with 2-D DIGE. Using liquid-phase IEF, the normal and leukemic cells were pre-fractionated into five subproteomes after multiplexing but prior to DIGE. Under these conditions, 2-D DIGE resolved >5000 protein-containing spots within the pH range 4.6-7.0. Differential labeling combined with subsequent MALDI-MS/MS identified proteins that were differentially expressed in leukemic cells. This analysis mapped protein identities to 128 mesenchymal progenitor cell proteins with at least one unique peptide match at >95% confidence. Of these proteins, 72 (56%) were expressed more than 1.25-fold higher or lower in leukemic cells compared with normal cells (p<0.05). These data were used to infer gene ontology biological processes that may be altered in leukemic bone marrow mesenchymal progenitor cells.     

Shotgun proteomic analysis of Bombyx mori brain: emphasis on regulation of behavior and development of the nervous system

The insect brain plays crucial roles in the regulation of growth and development and in all types of behavior. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and high‐performance liquid chromatography ? electron spray ionization tandem mass spectrometry (ESI‐MS/MS) shotgun to identify the proteome of the silkworm brain, to investigate its protein composition and to understand their biological functions. A total of 2210 proteins with molecular weights in the range of 5.64–1539.82 kDa and isoelectric points in the range of 3.78–12.55 were identified. These proteins were annotated according to Gene Ontology Annotation into the categories of molecular function, biological process and cellular component. We characterized two categories of proteins: one includes behavior‐related proteins involved in the regulation of behaviors, such as locomotion, reproduction and learning; the other consists of proteins related to the development or function of the nervous system. The identified proteins were classified into 283 different pathways according to KEGG analysis, including the PI3K‐Akt signaling pathway which plays a crucial role in mediating survival signals in a wide range of neuronal cell types. This extensive protein profile provides a basis for further understanding of the physiological functions in the silkworm brain.     

Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis

This work was performed to compare three precipitation protocols of protein extraction for 2-DE proteomic analysis using Arabidopsis leaf tissue: TCA-acetone, phenol, and TCA-acetone-phenol. There were no statistically significant differences in protein yield between the three methods. Samples were subjected to 2-DE in the 5 to 8 pH and 14-80 kDa ranges. The TCA-acetone-phenol protocol provided the best results in terms of spot focusing, resolved spots, spot intensity, unique spots detected, and reproducibility. In all, 93 qualitative or quantitative statistically significant differential spots were found between the three protocols. The 2-DE map of TCA-acetone-phenol extracts presented more resolved spots above 40 kDa, with no pI-dependent differences observed between the three protocols. 54 spots were selected for trypsin digestion, and the peptides were analyzed by MALDI-TOF-TOF MS. After database search using peptide mass fingerprinting, and MS/MS combined search, 30 proteins were identified, the proteins from chloroplastic photosynthetic and carbohydrate metabolism being those most highly represented. From these data, we were able to conclude that each extraction protocol had its main features. Considering this, the workflow of any standard comparative proteomic experiment should include the optimization and adaptation of the protein extraction protocol to the plant tissue and to the particular objective pursued.     

Analysis of Streptococcus mutans proteins modulated by culture under acidic conditions

Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.     

Studies of variability in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) through acorn protein profile analysis

Studies of variability in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.), the dominant tree species in the typical Mediterranean forest, have been carried out by using electrophoresis-based proteomic analysis of acorns. Ten populations distributed throughout the Andalusia region have been surveyed. Acorns were sampled from individual trees and proteins extracted from seed flour by using the TCA-acetone precipitation protocol. Extracts were subjected to SDS-PAGE and 2-DE for protein separation, gel images captured, spot or bands quantified, and subjected to statistical analysis (ANOVA, SOM and clustering). Variable bands or spots among populations were subjected to MALDI-TOF/TOF and LC-MS/MS for identification. The protein yield of the used protocol varied among populations, and it was in the 2.92-5.92 mg/g dry weight range. A total of 23 bands were resolved by SDS-PAGE in the 3-35 kDa Mr range, with 8 and 12, out of the total, showing respectively qualitative and quantitative statistically significant differences among populations. Data allowed grouping populations, with groups being correlated according to geographical location and climate conditions, to northern and southern, as well as the discrimination of both mesic and xeric groups. Acorn flour extracts from the most distant populations were analyzed by 2-DE, and 56 differential spots were proposed as markers of variability. Identified proteins were classified into two principal categories; storage and stress/defense protein. Besides providing the first reference map of mature acorn seeds, the use of SDS-PAGE and proteomics in characterizing natural biodiversity in forest trees will be discussed.     

Proteome analysis of the silkworm (Bombyx mori. L) colleterial gland during different development stages

The silkworm, Bombyx mori, colleterial gland developed very slowly until 2 days before emergence, then markedly enlarged due to the accumulation of a glue-like substances (mainly including 85% water and 11% proteins). However, the No glue (Ng) mutant female moth secreted only very little glue-like substance and laid loose eggs naturally. High-resolution two-dimensional polyacrylamide gel electrophoresis, followed by computer-assisted analysis, was used to screen the secretory region of colleterial gland protein patterns during different development stages to find quantitative and qualitative difference in protein expression during the pupae and moth stages. More than 700 protein spots were resolved in different developmental stages from the secretory region of the glands and most of the proteins were distributed in the mass range from 30 to 70 kD with pH 4-8. Through comparison and analysis, it was found that 3 proteins were only expressed in the later pupae stage (one or two days before emergence) and moth stage. Furthermore, these proteins were not expressed in the Ng mutant especially actin. There was a great variation of some protein expression volume during the development. Protein spots that changed more than 1.5-fold in expression level (relative to day 9), including 6 spots that were down-regulated and 2 spots that were up-regulated in expression were excised for identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Results indicated that actins that participated or regulated the exocytosis of colleterial gland and other differentially expressed proteins might be related to colleterial gland development or the secretion of a glue-like substance.