Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics

Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14(+) blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that approximately 40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca(2+) binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.     

Sequence of formation of molecular forms of plasminogen and plasmin-inhibitor complexes in plasma activated by urokinase or tissue-type plasminogen activator.

Total cellular polyadenylated RNA [poly(A)+ RNA] was prepared after guanidinium thiocyanate extraction of frozen brain tissue from age-matched normal and Down's-syndrome (trisomy 21) human foetuses. Poly(A)+ RNA populations were analysed by translation in vitro, followed by two-dimensional gel analysis by using both isoelectric focusing (ISODALT system) and non-equilibrium pH-gradient electrophoresis (BASODALT system) as the first-dimension separation. The relative concentrations of poly(A)+ RNA species coding for seven translation products were significantly altered in Down's syndrome, as determined by both visual comparisons of translation-product fluorograms from normal and Down's-syndrome samples and by quantitative radioactivity determination of individual translation products. The relative concentrations of mRNA species coding for two proteins (68 kDa and 49 kDa) were increased in Down's syndrome and may represent genes located on chromosome 21. The relative concentrations of mRNA species coding for five proteins (37 kDa, 35 kDa, 25.5 kDa, 24.5 kDa, 23 kDa) were decreased in Down's syndrome, these probably representing secondary effects of the trisomy. Six Down's-syndrome-linked translation products (49 kDa, 37 kDa, 33 kDa, 25.5 kDa, 24.5 kDa, 23 kDa) did not migrate with appreciable amounts of cellular proteins on two-dimensional gels and hence may represent either proteins of high turnover rates or those that are post-translationally modified in vivo. One translation product (68 kDa) comigrated with a major cellular protein species, which was identified as a 68 kDa microtubule-associated protein by limited peptide mapping. The significance of these changes is discussed in relation to the mechanisms whereby the Down's-syndrome phenotype is expressed in the human brain.     

Rice proteomics: a step toward functional analysis of the rice genome

The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms and/or expression that occurs under stresses. In this study, the catalogues of the rice proteome were constructed, and a functional characterization of some of these proteins was examined. Proteins extracted from tissues of rice and proteins extracted from rice under various kinds of stress were separated by two-dimensional PAGE. An image analyzer was used to reveal a total of 10,589 protein spots on 10 kinds of two-dimensional PAGE gels stained by Coomassie Brilliant Blue. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The internal amino acid sequences of 633 proteins were determined using a protein sequencer or mass spectrometry after enzyme digestion of the proteins. Finally, a data file of rice proteins that included information on amino acid sequences and sequence homologies was constructed. The major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that turned out to be calreticulin and a gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have functions in the signal transduction pathway. The information thus obtained from the rice proteome will be helpful in predicting the function of the unknown proteins and will aid in their molecular cloning.     

Isolation and analysis of a fur mutant of Neisseria gonorrhoeae.

The pathogenic Neisseria spp. produce a number of iron-regulated gene products that are thought to be important in virulence. Iron-responsive regulation of these gene products has been attributed to the presence in Neisseria spp. of the Fur (ferric uptake regulation) protein. Evidence for the role of Fur in neisserial iron regulation has been indirect because of the inability to make fur null mutations. To circumvent this problem, we used manganese selection to isolate missense mutations of Neisseria gonorrhoeae fur. We show that a mutation in gonococcal fur resulted in reduced modulation of expression of four well-studied iron-repressed genes and affected the iron regulation of a broad range of other genes as judged by two-dimensional polyacrylamide gel electrophoresis (PAGE). All 15 of the iron-repressed spots observed by two-dimensional PAGE were at least partially derepressed in the fur mutant, and 17 of the 45 iron-induced spots were affected by the fur mutation. Thus, Fur plays a central role in regulation of iron-repressed gonococcal genes and appears to be involved in regulation of many iron-induced genes. The size and complexity of the iron regulons in N. gonorrhoeae are much greater than previously recognized.     

Proteomic analysis of lymph

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), two-dimensional gel electrophoresis (2-D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI-TOF-MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2-D PAGE analysis. MS analysis of a large number of protein spots from 2-D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2-D PAGE gels of lymph included, fibrinogen alpha- and beta-chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A-1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor-1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.     

Protein expression analysis of Halobacillus dabanensis D-8T subjected to salt shock

To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis D-8T. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain D-8T are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and Imagemaster 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, 8 proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.     

Differential protein expression in the cytosol fraction of an MCF-7 breast cancer cell line selected for resistance toward melphalan

Analysis of differential protein expression in the cytosol of melphalan-resistant and -susceptible MCF-7 cell lines has been carried out using a combination of two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics. Comparison of multiple digitized gel arrays detected several spots as candidates for differentially expressed proteins in melphalan-resistant MCF-7 cells. The up-regulated proteins included retinoic acid binding protein II, an isoform of the macrophage migration inhibition factor, and other unidentified proteins. The down-regulated proteins included calreticulin, cyclophin A, and an isoform of the 27 kD heat shock protein. Correlation of the differential expression of some of the proteins with acquired resistance of MCF7 cells to melphalan is discussed.     

Ribonucleic Acid and protein metabolism in pea epicotyls : I. The aging process

Aging of actively growing, etiolated pea Pisum sativum L. var Alaska plants was initiated by removing the plumules of plants in the third internode stage, and applying lanolin to the cut apices of otherwise intact plants. During the subsequent 4-day aging period, several degenerative events occurred in this apical 10-millimeter region. Ribosomal RNA and messenger RNA contents declined, polyribosomes disaggregated, and the protein synthesizing capacity of the polysomes decreased.

Two-dimensional, silver-stained protein patterns revealed that aging altered the relative amounts of specific cellular proteins accumulated in vivo. In addition, polypeptide patterns generated by cell-free translation of total and polysomal RNA, isolated from unaged and aged tissues, showed major modifications. More than 200 spots could be resolved by two-dimensional gel fluorography of translation products using RNA from fresh tissues. Of these 200 spots, about eight appeared or increased when total RNA from aged tissue was used, and about 58 disappeared or declined. When polysomal RNA from aged tissue was used as template, about 12 spots appeared or increased, whereas about 64 disappeared or decreased. In general, the products which increased after aging were lower molecular weight and those that decreased were higher molecular weight.

     

Towards an analysis of the rice mitochondrial proteome

Purified rice (Oryza sativa) mitochondrial proteins have been arrayed by isoelectric focusing/polyacrylamide gel electrophoresis (PAGE), by blue-native (BN) PAGE, and by reverse-phase high-performance liquid chromatography (LC) separation (LC-mass spectrometry [MS]). From these protein arrays, we have identified a range of rice mitochondrial proteins, including hydrophilic/hydrophobic proteins (grand average of hydropathicity = -1.27 to +0.84), highly basic and acid proteins (isoelectric point = 4.0-12.5), and proteins over a large molecular mass range (6.7-252 kD), using proteomic approaches. BN PAGE provided a detailed picture of electron transport chain protein complexes. A total of 232 protein spots from isoelectric focusing/PAGE and BN PAGE separations were excised, trypsin digested, and analyzed by tandem MS (MS/MS). Using this dataset, 149 of the protein spots (the products of 91 nonredundant genes) were identified by searching translated rice open reading frames from genomic sequence and six-frame translated rice expressed sequence tags. Sequence comparison allowed us to assign functions to a subset of 85 proteins, including many of the major function categories expected for this organelle. A further six spots were matched to rice sequences for which no specific function has yet been determined. Complete digestion of mitochondrial proteins with trypsin yielded a peptide mixture that was analyzed directly by reverse-phase LC via organic solvent elution from a C-18 column (LC-MS). These data yielded 170 MS/MS spectra that matched 72 sequence entries from open reading frame and expressed sequence tag databases. Forty-five of these were obtained using LC-MS alone, whereas 28 proteins were identified by both LC-MS and gel-based separations. In total, 136 nonredundant rice proteins were identified, including a new set of 23 proteins of unknown function located in plant mitochondria. We also report the first direct identification, to our knowledge, of PPR (pentatricopeptide repeat) proteins in the plant mitochondrial proteome. This dataset provides the first extensive picture, to our knowledge, of mitochondrial functions in a model monocot plant.     

Proteome analysis of an aerobic hyperthermophilic crenarchaeon, Aeropyrum pernix K1

We analyzed the proteome of a crenararchaeon, Aeropyrum pernix K1, by using the following four methods: (i) two-dimensional PAGE followed by MALDI-TOF MS, (ii) one-dimensional SDS-PAGE in combination with two-dimensional LC-MS/MS, (iii) multidimensional LC-MS/MS, and (iv) two-dimensional PAGE followed by amino-terminal amino acid sequencing. These methods were found to be complementary to each other, and biases in the data obtained in one method could largely be compensated by the data obtained in the other methods. Consequently a total of 704 proteins were successfully identified, 134 of which were unique to A. pernix K1, and 19 were not described previously in the genomic annotation. We found that the original annotation of the genomic data of this archaeon was not adequate in particular with respect to proteins of 10-20 kDa in size, many of which were described as hypothetical. Furthermore the amino-terminal amino acid sequence analysis indicated that surprisingly the translation of 52% of their genes starts with TTG in contrast to ATG (28%) and GTG (20%). Thus, A. pernix K1 is the first example of an organism in which TTG is the most predominant translational initiation codon.