A Sampling of the Yeast Proteome

In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.     

Electrophoretic analysis of the cytoplasmic and nuclear protein changes after induction of differentiation in WEHI-3B myelomonocytic leukemia cells

In response to a differentiation factor (G-CSF) the myelomonocytic leukemia cell line (WEHI-3B(D+) differentiates to form mature macrophages and neutrophils. The effect of G-CSF on WEHI-3B(D+) differentiation was augmented by low concentrations (5 ng/ml) of actinomycin D. Quantitative binding of an antineutrophil serum was used to segregate the differentiated cells from the leukemic blast cells. Molecular markers of later myeloid differentiation were detected in myelocytes and macrophages purified from differentiating WEHI-3B(D+) cells. To study the initial molecular processes associated with the initiation of WEHI-3B(D+) cells to differentiation, the protein changes were analyzed using gel electrophoresis. Quantitative analysis of the fluorographs from the two-dimensional (2D) electrophorograms of the 35S-labeled proteins revealed major changes in the biosynthetic rates for 16 proteins within 5 hr: The biosynthesis of six proteins was increased and another ten proteins were synthesized at a reduced rate. Two of the proteins (17K and 36K daltons) were located in the nucleus. Pulse-chase experiments indicated that protein turnover for these proteins was rapid but the degradation of four proteins was suppressed. At least six of the proteins (16K to 120K daltons) were acidic and were associated with the cytoplasm. Electrophoretic analysis of the 35S-labeled proteins indicated that a 35K protein induced by G-CSF was found in high abundance only in purified cells of intermediate differentiation (eg, myelocytes). Other proteins (eg, a very high molecular weight protein, and a 16K dalton protein) were obviously late markers of differentiated neutrophils or macrophages.     

Regulation of initiation factors during translational repression caused by serum depletion. Abundance, synthesis, and turnover rates

During growth in unreplenished medium, the fraction of active, polysomal ribosomes progressively decreases about 3-fold from 80-90% to only 20-40% due to a reduced rate of initiation. To assess whether the abundance of initiation factors could be involved in this repression of translational activity. HeLa cell cytoplasmic lysates were resolved by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and spots corresponding to the initiation factor proteins were quantitated. The relative abundance of most of the initiation factor proteins only decreases by 10-40% and roughly parallels that of the ribosomes. Measurement of the rates of synthesis and turnover of the initiation factor proteins establishes that during periods of active growth, synthesis and degradation occur coordinately with total cell protein. As growth rate decreases, the synthesis of some initiation factor proteins, particularly eukaryotic initiation factor (eIF)-3 subunits, becomes depressed. Serum stimulation of serum-depleted cells recruits most inactive ribosomes and mRNAs into polysomes, but most initiation factor mRNAs are not selectively recruited. The principal exceptions are eIF-3p24 which exhibits 4-5 fold enhanced synthesis and eIF-3p44 and eIF-4A whose syntheses are moderately stimulated.     

Study of early leaf senescence in Arabidopsis thaliana by quantitative proteomics using reciprocal 14N/15N labeling and difference gel electrophoresis

Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy simultaneously employing fluorescent two-dimensional DIGE as well as metabolic (15)N labeling followed by MS. Reciprocal (14)N/(15)N labeling of entire Arabidopsis thaliana plants showed that full incorporation of (15)N into the proteins of the plant did not cause any adverse effects on development and protein expression. A direct comparison of DIGE and (15)N labeling combined with MS showed that results obtained by both quantification methods correlated well for proteins showing low to moderate regulation factors. Nano HPLC/ESI-MS/MS analysis of 21 protein spots that consistently exhibited abundance differences in nine biological replicates based on both DIGE and MS resulted in the identification of 13 distinct proteins and protein subunits that showed significant regulation in Arabidopsis mutant plants displaying advanced leaf senescence. Ribulose 1,5-bisphosphate carboxylase/oxygenase large and three of its four small subunits were found to be down-regulated, which reflects the degradation of the photosynthetic machinery during leaf senescence. Among the proteins showing higher abundance in mutant plants were several members of the glutathione S-transferase family class phi and quinone reductase. Up-regulation of these proteins fits well into the context of leaf senescence since they are generally involved in the protection of plant cells against reactive oxygen species which are increasingly generated by lipid degradation during leaf senescence. With the exception of one glutathione S-transferase isoform, none of these proteins has been linked to leaf senescence before.     

Initial analysis of the phosphoproteome of Chinese hamster ovary cells using electrophoresis.

Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis. Chinese hamster ovary (CHO) cells were metabolically labeled using 32P-orthophosphate. The proteins were extracted and run on two-dimensional electrophoresis. Gels were stained using colloidal Coomassie stain, dried, and phosphorimaged. The Coomassie stain allowed the observation of 468 individual protein spots. The phosphorimage showed 181 spots. The phosphoproteome of CHO cells therefore comprises around one third as many proteins as the CHO cell abundance proteome. However, the most intense spots in the phosphoproteome usually do not correlate with intense spots in the abundance proteome. We investigated the effects of labeling time, finding that the number of observable spots increases but the relative intensities also change. We also investigated the effects of adding a phosphatase inhibitor during labeling. Finally, we evaluated a phosphoprotein-specific stain (Pro-Q Diamond) in comparison with radiolabeling methods. There is not perfect correlation between radiolabeled phosphoproteins and Pro-Q Diamond-stained phosphoproteins.     

Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1

Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes trade mark and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder trade mark software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10(-9)) were identified. For comparison, another experiment was carried out with four SYPRO-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster trade mark 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 microg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.     

Cytochalasin D alters the rate of synthesis of some HEp-2 cytoskeletal proteins. Examination by two-dimensional gel electrophoresis

The most abundant proteins of HEp-2 cells were resolved by two-dimensional gel electrophoresis. The protein spots corresponding to several cytoskeletal proteins (vimentin, alpha-tubulin, beta-tubulin, alpha-actinin, tropomyosins, and cytokeratins) were identified by comigration with protein markers or by immunological techniques. After treatment of HEp-2 cells with 0.2 microM or 2.0 microM cytochalasin D for 20 h, radioautograms of two-dimensional gel patterns of lysates from cells pulse-labeled with [35S]methionine indicated that the drug altered the rate of synthesis of some proteins. The relative rate of synthesis of the identified cytoskeletal proteins was measured. Synthesis of alpha-actinin, the higher-molecular-mass pair of tropomyosins and actin were similarly increased with cytochalasin D treatment, suggesting coordinate induction. Vimentin and tubulin synthesis was depressed. One cytokeratin exhibited an increase in synthesis comparable to actin, another was increased to a lesser extent and one was decreased.     

Proteomics analysis of date palm leaves affected at three characteristic stages of brittle leaf disease

Proteomics analysis has been performed in leaf tissue from field date palm trees showing the brittle leaf disease (BLD) or maladie des feuilles cassantes, the main causal agent of the date palm decline in south Tunisia. To study the evolution of the disease, proteins from healthy and affected leaves taken at three disease stages (S1, S2 and S3) were trichloroacetic acid acetone extracted and subjected to two-dimensional gel electrophoresis (5–8 pH range). Statistical analysis showed that the protein abundance profile is different enough to differentiate the affected leaves from the healthy ones. Fifty-eight variable spots were successfully identified by matrix-assisted laser desorption ionization time of flight, 60 % of which corresponded to chloroplastic ones being involved in the photosynthesis electronic chain and ATP synthesis, metabolic pathways implicated in the balance of the energy, and proteases. Changes in the proteome start at early disease stage (S1), and are greatest at S2. In addition to the degradation of the ribulose-1.5-bisphosphate carboxylase oxygenase in affected leaflets, proteins belonging to the photosynthesis electronic chain and ATP synthesis decreased following the disease, reinforcing the relationship between BLD and manganese deficiency. The manganese-stabilizing proteins 33 kDa, identified in the present work, can be considered as protein biomarkers of the disease, especially at early disease step.     

Proteomic analysis of cold stress-responsive proteins in <Emphasis Type="Italic">Thellungiella</Emphasis> rosette leaves

Low temperature is one of the most severe environmental factors that impair plant growth and agricultural production. To investigate how Thellungiella halophila, an Arabidopsis-like extremophile, adapts to cold stress, a comparative proteomic approach based on two-dimensional electrophoresis was adopted to identify proteins that changed in abundance in Thellungiella rosette leaves during short term (6 h, 2 and 5 days) and long term (24 days) exposure to cold stress. Sixty-six protein spots exhibited significant change at least at one time point and maximal cold stress induced-proteome change was found in long-term cold stress group while the minimal change was found in 6-h cold treatment group. Fifty protein spots were identified by mass spectrometry analysis. The identified proteins mainly participate in photosynthesis, RNA metabolism, defense response, energy pathway, protein synthesis, folding and degradation, cell wall and cytoskeleton and signal transduction. These proteins might work cooperatively to establish a new homeostasis under cold stress. Nearly half of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology suggesting that the cold stress tolerance of T. halophila is achieved, at least partly, by regulation of chloroplast function. All protein spots involved in RNA metabolism, defense response, protein synthesis, folding and degradation were found to be upregulated markedly by cold treatment, indicating enhanced RNA metabolism, defense and protein metabolism may play crucial roles in cold tolerance mechanism in T. halophila.     

Proteomic analysis of doxorubicin‐induced changes in the proteome of HepG2cells combining 2‐D DIGE and LC‐MS/MS approaches

HepG‐2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2‐D gel‐based and gel‐free methods. The analysis of crude HepG2 cell extracts by 2‐D DIGE provided data on 1835 protein spots which was then complemented by MS‐centered analysis of stable isotope labeling by amino acids in cell culture‐labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin‐induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin‐associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.     

Identification of the major membrane and core proteins of vaccinia virus by two-dimensional electrophoresis.

Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known about the molecular events that occur during that process. Towards this goal, we have identified the major membrane and core proteins of the intracellular mature virus (IMV). Pure IMV preparations were subjected to Nonidet P-40 (NP-40) and dithiothreitol (DTT) treatment to separate the core proteins from the membrane proteins. These proteins were subsequently separated by two-dimensional (2D) gel electrophoresis, and the major polypeptide spots, as detected by silver staining and 35S labeling, were identified by either matrix-assisted laser desorption/ionization mass spectrometry, N-terminal amino acid sequencing, or immunoprecipitation with defined antibodies. Sixteen major spots that partitioned into the NP-40-DTT-soluble fraction were identified; 11 of these were previously described virally encoded proteins and 5 were cellular proteins, mostly of mitochondrial origin. The core fraction revealed four major spots of previously described core proteins, two of which were also detected in the membrane fraction. Subsequently, the NP-40-DTT-soluble and -insoluble fractions from purified virus preparations, separated by 2D gels, were compared with postnuclear supernatants of infected cells that had been metabolically labeled at late times (6 to 8 h) postinfection. This relatively short labeling period as well as the apparent shutoff of host protein synthesis allowed the selective detection in such postnuclear supernatants of virus-encoded proteins. These postnuclear supernatants were subsequently treated with Triton X-114 or with sodium carbonate to distinguish the membrane proteins from the soluble proteins. We have identified the major late membrane and nonmembrane proteins of the IMV as they occur in the virus as well as in infected cells. This 2D gel map should provide an important reference for future molecular studies of vaccinia virus morphogenesis.     

Use of a proteome strategy for tagging proteins present at the plasma membrane

A plasma membrane (PM) fraction was purified from Arabidopsis thaliana using a standard procedure and analyzed by two-dimensional (2D) gel electrophoresis. The proteins were classified according to their relative abundance in PM or cell membrane supernatant fractions. Eighty-two of the 700 spots detected on the PM 2D gels were microsequenced. More than half showed sequence similarity to proteins of known function. Of these, all the spots in the PM-specific and PM-enriched fractions, together with half of the spots with similar abundance in PM fraction and supernatant, have previously been found at the PM, supporting the validity of this approach. Extrapolation from this analysis indicates that (i) approximately 550 polypeptides found at the PM could be resolved on 2D gels; (ii) that numerous proteins with multiple locations are found at the PM; and (iii) that approximately 80% of PM-specific spots correspond to proteins with unknown function. Among the later, half are represented by ESTs or cDNAs in databases. In this way, several unknown gene products were potentially localized to the PM. These data are discussed with respect to the efficiency of organelle proteome approaches to link systematically genomic data to genome expression. It is concluded that generalized proteomes can constitute a powerful resource, with future completion of Arabidopsis genome sequencing, for genome-wide exploration of plant function.     

Impact of oxidative stress on the function,abundance, and turnover of the Arabidopsis 80S cytosolic ribosome

Abiotic stress in plants causes accumulation of reactive oxygen species (ROS) leading to the need for new protein synthesis to defend against ROS and to replace existing proteins that are damaged by oxidation. Functional plant ribosomes are critical for these activities, however we know little about the impact of oxidative stress on plant ribosome abundance, turnover, and function. Using Arabidopsis cell culture as a model system, we induced oxidative stress using 1 µm of H₂O₂ or 5 µm menadione to more than halve cell growth rate and limit total protein content. We show that ribosome content on a total cell protein basis decreased in oxidatively stressed cells. However, overall protein synthesis rates on a ribosome abundance basis showed the resident ribosomes retained their function in oxidatively stressed cells. ¹⁵N progressive labelling was used to calculate the rate of ribosome synthesis and degradation to track the fate of 62 r‐proteins. The degradation rates and the synthesis rates of most r‐proteins slowed following oxidative stress leading to an ageing population of ribosomes in stressed cells. However, there were exceptions to this trend; r‐protein RPS14C doubled its degradation rate in both oxidative treatments. Overall, we show that ribosome abundance decreases and their age increases with oxidative stress in line with loss of cell growth rate and total cellular protein amount, but ribosome function of the ageing ribosomes appeared to be maintained concomittently with differences in the turnover rate and abundance of specific ribosomal proteins. Data are available via ProteomeXchange with identifier PXD012840.     

The Fas-induced apoptosis analyzed by high throughput proteome analysis

The fate of cytosolic proteins was studied during Fas-induced cell death of Jurkat T-lymphocytes by proteome analysis. Among 1000 spots resolved in two-dimensional gels, comparison of control versus apoptotic cells revealed that the signal intensity of 19 spots decreased or even disappeared, whereas 38 novel spots emerged. These proteins were further analyzed with respect to de novo protein synthesis, phosphorylation status, and intracellular localization by metabolic labeling and analysis of subcellular protein fractions in combination with two-dimensional Western blots and mass spectrometry analysis of tryptic digests. We found that e.g. hsp27, hsp70B, calmodulin, and H-ras synthesis was induced upon Fas signaling. 34 proteins were affected by dephosphorylation (e.g. endoplasmin) and phosphorylation (e.g. hsc70, hsp57, and hsp90). Nuclear annexin IV translocated to the cytosol, whereas decreasing cytosolic TCP-1alpha became detectable in the nucleus. In addition, degradation of 12 proteins was observed; among them myosin heavy chain was identified as a novel caspase target. Fas-induced proteome alterations were compared with those of other cell death inducers, indicating specific physiological characteristics of different cell death mechanisms, consequent to as well as independent of caspase activation. Characteristic proteome alterations of apoptotic cells at early time points were found reminiscent of those of malignant cells in vivo.     

Photolabeling of protein components in the pactamycin binding site of rat liver ribosomes

The antitumoral and antibacterial drug pactamycin can be radioactively labeled by iodination without loss of biological activity. Using the labeled pactamycin, the ribosomal binding site of the drug on rat liver ribosomes has been studied by affinity labeling techniques taking advantage of the photoreactive acetophenone group present in the molecule. When 40 S ribosomal subunits are labeled, one major spot of radioactivity is found associated to protein S25. In addition, weaker spots related to proteins S14/15, S10, S17 and S7 can also be detected in the autoradiogram of the two-dimensional gel slab. Since pactamycin inhibits protein synthesis initiation, the proteins forming its binding site must be related to some step of this process. By comparison with results from pactamycin affinity labeling of Escherichia coli ribosomes (Tejedor, F., Amils, R. and Ballesta, J.P.G. (1985) Biochemistry 24, 3667-3672) these proteins could lie in the mRNA and initiation factors binding region of the rat liver ribosome.     

Exchange and stability of HeLa ribosomal proteins in vivo.

The relative stabilities of individual HeLa ribosomal proteins and their capacity for exchange between ribosome-bound and -free states in the cytoplasm were examined. Most ribosomal proteins on cytoplasmic ribosomes were found to have uniform, high stability as measured by comparing the short term (12-hour) to steady state (3-day) labeling ratios determined for each ribosomal protein. This would be expected if the proteins in ribosomes either were all stable or were all degraded as a unit. The data do not rule out the possibility that individual proteins have different stabilities prior to their assembly into ribosomes. Four proteins labeled atypically. One large subunit protein (L5) had a lower than average ratio. We interpret this low ratio as being due to a large free pool of this protein. Three proteins (L10, L28, S2) had higher than average ratios, interpreted as being due to reduced protein stability. Two of these proteins (L10, L28) with high ratios were also found to exchange in vivo. The exchangeable proteins may be subject to increased degradation during the time that they spend in the exchangeable free pool. The third protein (S2) with an atypically high ratio is thought to be degraded or altered while on the ribosome, or slowly lost as ribosomes age, because exchange of this protein was not detected. These interpretations and some alternate interpretations are explained. The exchange of three large subunit proteins (L10, L19, L28) was detected by labeling of protein after ribosome synthesis had been inhibited with actinomycin D. Autoradiography of two-dimensional polyacrylamide gels showed labeling of these spots.