A comprehensive proteome map of the lipid-requiring nosocomial pathogen Corynebacterium jeikeium K411

Corynebacterium jeikeium is a lipid-requiring pathogen that is considered as part of the normal microflora of the human skin and associated with severe nosocomial infections. Systematic reference maps of the cytoplasmic, cell surface-associated, and extracellular proteome fractions of the clinical isolate C. jeikeium K411 were examined by 2-DE coupled with MALDI-TOF MS. A sum total of 555 protein spots were identified by PMF, corresponding to 358 different proteins that were classified into functional categories and integrated into metabolic pathways. The majority of the proteins were linked to housekeeping functions in energy production and translation and to physiological processes in amino acid, carbohydrate, nucleotide, and lipid metabolism. A complete enzymatic machinery necessary to utilize exogenous fatty acids by beta-oxidation was detected in the cytoplasmic proteome fraction. In addition, several predicted virulence factors of C. jeikeium K411 were identified in the cell surface-associated and extracellular subproteome, including the cell surface proteins SurA and SurB, the surface-anchored pilus subunits SapA and SapB, the surface-anchored collagen adhesin CbpA, the cholesterol esterase Che, and the acid phosphatase AcpA.     

Cell wall proteins in apoplastic fluids of Arabidopsis thaliana rosettes: identification by mass spectrometry and bioinformatics

Weakly bound cell wall proteins of Arabidopsis thaliana were identified using a proteomic and bioinformatic approach. An efficient protocol of extraction based on vacuum-infiltration of the tissues was developed. Several salts and a chelating agent were compared for their ability to extract cell wall proteins without releasing cytoplasmic contaminants. Of the 93 proteins that were identified, a large proportion (60%) was released by calcium chloride. From bioinformatics analysis, it may be predicted that most of them (87 out of 93) had a signal peptide, whereas only six originated from the cytoplasm. Among the putative apoplastic proteins, a high proportion (67 out of 87) had a basic pI. Numerous glycoside hydrolases and proteins with interacting domains were identified, in agreement with the expected role of the extracellular matrix in polysaccharide metabolism and recognition phenomena. Ten proteinases were also found as well as six proteins with unknown functions. Comparison of the cell wall proteome of rosettes with the previously published cell wall proteome of cell suspension cultures showed a high level of cell specificity, especially for the different members of several large multigenic families.     

Frozen tissue can provide reproducible proteomic results of subcellular fractionation

Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography–tandem mass spectrometry (LC–MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SI_N) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SI_N values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method.     

Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins.     

The human erythrocyte proteome: analysis by ion trap mass spectrometry

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, "spectrin extract" mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined.     

Proteomic approach to study leaf proteins in a fast-growing tree species, Gmelina arborea Linn. Roxb

Foliar proteome studies have become highly significant for a comprehensive understanding of complex processes associated with plant growth and development. In the present study, we present a proteomic approach to analyze leaf proteins in an important timber-yielding and fast-growing forest tree species, Gmelina arborea Linn. Roxb. (Verbanaceae). Foliar protein analysis involved protein extraction, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight (MALDI–TOF–TOF). From the 2-DE protein profile of Gmelina leaves, we identified and isolated 150 well-separated protein spots; among these, 64 protein spots were identified by mass spectrometric (MS/MS) analysis. These proteins were classified according to their involvement in basic biological functions, such as photosynthesis, amino acid metabolism, cytoskeleton, cell wall metabolism, stress-related proteins, redox maintenance, electron transport chain, phytohormone metabolism and protein translation and folding. Analytical variance was determined for the protein spots of samples from different plants. The present study is believed to provide a foundation for the use of leaf proteomics in addressing fundamental physiological and biochemical processes associated with growth and productivity of tree species such as Gmelina arborea.     

Proteomics of ionically bound and soluble extracellular proteins in Medicago truncatula leaves

A large proportion of the apoplast proteome resides in the intercellular fluid (IF) or is ionically bound (IB) to the wall matrix. A combined analysis of IF and IB proteins of the Medicago truncatula leaf apoplast was performed. 2-DE analyses demonstrated the reproducible presence of 220 IF and 84 IB proteins in the apoplast. These two protein populations were largely distinct; 22 proteins could be spatially matched, but MALDI-TOF/TOF analyses suggested a considerably smaller number had common identities. MALDI-TOF/TOF characterisation identified 81 distinct proteins. Analyses of selected IF proteins (45) indicated 17 distinct proteins with mainly defence-related functions, whereas analyses of IB proteins (70) identified 63 distinct proteins of diverse natures, including proteins of non-canonical natures. The presence of non-canonical proteins in IB extracts is discussed in the light of evidence supporting a low level of contamination of purified walls from symplastic proteins. This work indicates that IB and IF proteins are functionally distinct fractions of the apoplast. The data obtained complements earlier studies of the Medicago proteome and therefore will be useful in future studies investigating the role of apoplastic proteins in plant processes.     

Analysis of cell wall material from plant tissues: Extraction and purification

The alcohol-insoluble residue (AIR) of immature and mature runner beans contains co-precipitated cytoplasmic proteins, nucleic acids, starch and polyphenols, which contaminate the isolated polysaccharide fractions and their binding is sufficiently tenacious to resist complete extraction with the usual protein solubilizing reagents. Therefore, a method was developed for preparation of “cell wall material” from plant tissues in which the contamination with cytoplasmic constituents was minimal. Alternative solvents for cell disruption and protein extraction have been compared. The method depended for its success on the selective removal of the contaminants from fresh ball-milled tissue by sequential treatments with 1% aq. Na deoxycholate, PhOH-HOAc-H₂O followed by α-amylase digestion. Ball-milling the tissue ensured almost complete rupture of the cells and organelles and allowed the solvents to penetrate the sample fully and dissolve the cytoplasmic constituents. The purified “cell wall material” has protein contents varying from 2.5 to 5.5% depending on the type and maturity of the tissue. The residual proteins are resistant to pronase, rich in hydroxyproline and have the amino acid composition of purified cell wall proteins, showing that the wall preparations are relatively pure.     

A role for BELLRINGER in cell wall development is supported by loss-of-function phenotypes

Studies reported unintended pleiotropic effects for a number of pesticidal proteins ectopically expressed in transgenic crops, but the nature and significance of such effects in planta remain poorly understood. Here we assessed the effects of corn cystatin II (CCII), a potent inhibitor of C1A cysteine (Cys) proteases considered for insect and pathogen control, on the leaf proteome and pathogen resistance status of potato lines constitutively expressing this protein. The leaf proteome of lines accumulating CCII at different levels was resolved by 2-dimensional gel electrophoresis and compared with the leaf proteome of a control (parental) line. Out of ca. 700 proteins monitored on 2-D gels, 23 were significantly up- or downregulated in CCII-expressing leaves, including 14 proteins detected de novo or up-regulated by more than five-fold compared to the control. Most up-regulated proteins were abiotic or biotic stress-responsive proteins, including different secretory peroxidases, wound inducible protease inhibitors and pathogenesis-related proteins. Accordingly, infection of leaf tissues by the fungal necrotroph Botryris cinerea was prevented in CCII-expressing plants, despite a null impact of CCII on growth of this pathogen and the absence of extracellular Cys protease targets for the inhibitor. These data point to the onset of pleiotropic effects altering the leaf proteome in transgenic plants expressing recombinant protease inhibitors. They also show the potential of these proteins as ectopic modulators of stress responses in planta, useful to engineer biotic or abiotic stress tolerance in crop plants of economic significance.     

Analysis of grape berry cell wall proteome: a comparative evaluation of extraction methods

Different methods were tested for the extraction of proteins from the cell wall-enriched fraction (CWEf) obtained from a sample formed by skin and seeds of ripe berries of Vitis vinifera L. cv. Cabernet Sauvignon. The CWEf was isolated using a disruptive approach that involves tissue homogenization and precipitation by centrifugation. To extract proteins, the CWEf was treated with CaCl(2) and LiCl in two successive steps or, alternatively, with phenol. The efficiency of the protocols was evaluated by measuring protein yield and by analyzing two-dimensional gel electrophoresis (2-DE) gels for the highest detectable spot number and the greatest spot resolution. The phenol method was also adopted for the extraction of proteins from the cytosolic fraction (CYf). The comparison of 2-DE reference maps of protein extracts from CWEf and CYf indicated the presence of both common traits and unique characteristics. To survey this aspect some spots detected in both fractions or present in only one fraction were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Of the 47 spots identified, some were found to be cell wall proteins, while others were proteins not traditionally considered as localized in the apoplastic space. The data presented here provide initial information regarding the apoplastic proteome of grape berry tissues, but also raise the issue of the technical problems that characterize the isolation of cell wall proteins from these very hardy tissues.     

Proteomics in Trypanosoma cruzi--localization of novel proteins to various organelles

The completion of the genome sequence of Trypanosoma cruzi has been followed by several studies of protein expression, with the long-term aim to obtain a complete picture of the parasite proteome. We report a proteomic analysis of an organellar cell fraction from T. cruzi CL Brener epimastigotes. A total of 396 proteins were identified by LC-MS/MS. Of these, 138 were annotated as hypothetical in the genome databases and the rest could be assigned to several metabolic and biosynthetic pathways, transport, and structural functions. Comparative analysis with a whole cell proteome study resulted in the validation of the expression of 173 additional proteins. Of these, 38 proteins previously reported in other stages were not found in the only large-scale study of the total epimastigote stage proteome. A selected set of identified proteins was analyzed further to investigate gene copy number, sequence variation, transmembrane domains, and targeting signals. The genes were cloned and the proteins expressed with a c-myc epitope tag in T. cruzi epimastigotes. Immunofluorescence microscopy revealed the localization of these proteins in different cellular compartments such as ER, acidocalcisome, mitochondrion, and putative cytoplasmic transport or delivery vesicles. The results demonstrate that the use of enriched subcellular fractions allows the detection of T. cruzi proteins that are undetected by whole cell proteomic methods.     

Parallel Proteomic and Phosphoproteomic Analyses of Successive Stages of Maize Leaf Development

We performed large-scale, quantitative analyses of the maize (Zea mays) leaf proteome and phosphoproteome at four developmental stages. Exploiting the developmental gradient of maize leaves, we analyzed protein and phosphoprotein abundance as maize leaves transition from proliferative cell division to differentiation to cell expansion and compared these developing zones to one another and the mature leaf blade. Comparison of the proteomes and phosphoproteomes suggests a key role for posttranslational regulation in developmental transitions. Analysis of proteins with cell wall– and hormone-related functions illustrates the utility of the data set and provides further insight into maize leaf development. We compare phosphorylation sites identified here to those previously identified in Arabidopsis thaliana. We also discuss instances where comparison of phosphorylated and unmodified peptides from a particular protein indicates tissue-specific phosphorylation. For example, comparison of unmodified and phosphorylated forms of PINFORMED1 (PIN1) suggests a tissue-specific difference in phosphorylation, which correlates with changes in PIN1 polarization in epidermal cells during development. Together, our data provide insights into regulatory processes underlying maize leaf development and provide a community resource cataloging the abundance and phosphorylation status of thousands of maize proteins at four leaf developmental stages.     

Method for Label-Free Quantitative Proteomics for <Emphasis Type="Italic">Sorghum bicolor</Emphasis> L. Moench

Sorghum (Sorghum bicolor L. Moench) is a rapidly emerging high biomass feedstock for bioethanol and lignocellulosic biomass production. The robust varietal germplasm of sorghum and its completed genome sequence provide the necessary genetic and molecular tools to study and engineer the biotic/abiotic stress tolerance. Traditional proteomics approaches for outlining the sorghum proteome have many limitations like, demand for high protein amounts, reproducibility and identification of only few differential proteins. In this study, we report a gel-free, quantitative proteomic method for in-depth coverage of the sorghum proteome. This novel method combining phenol extraction and methanol chloroform precipitation gives high total protein yields for both mature sorghum root and leaf tissues. We demonstrate successful application of this method in comparing proteomes of contrasting cultivars of sorghum, at two different phenological stages. Protein identification and relative quantification analyses were performed by a label-free liquid chromatography tandem mass spectrometry (LC/MS-MS) analyses. Several unique proteins were identified respectively from sorghum tissues, specifically 271 from leaf and 774 from root tissues, with 193 proteins common in both tissues. Using gene ontology analysis, the differential proteins identified were finely corroborated with their leaf/root tissue specific functions. This method of protein extraction and analysis would contribute substantially to generate in-depth differential protein data in sorghum as well as related species. It would also increase the repertoire of methods uniquely suited for gel-free plant proteomics that are increasingly being developed for studying abiotic and biotic stress responses.     

Proteomic analysis of the secretome of rice calli

The cell wall and extracellular matrix in higher plants include secreted proteins that play critical roles in a wide range of cellular processes, such as structural integrity and biogenesis. Compared with the intensive cell wall proteomic studies in Arabidopsis , the list of cell wall proteins identified in monocot species is lacking. Therefore, we conducted a large-scale proteomic analysis of secreted proteins from rice. Highly purified secreted rice proteins were obtained from the medium of a suspension of callus culture and were analyzed with multidimensional protein identification technology (MudPIT). As a result, we could detect a total of 555 rice proteins by MudPIT analysis. Based on bioinformatic analyses, 27.7% (154 proteins) of the identified proteins are considered to be secreted proteins because they possess a signal peptide for the secretory pathway. Among the 154 identified proteins, 27% were functionally categorized as stress response proteins, followed by metabolic proteins (26%) and factors involved in protein modification (24%). Comparative analysis of cell wall proteins from Arabidopsis and rice revealed that one third of the secreted rice proteins overlapped with those of Arabidopsis . Furthermore, 25 novel rice-specific secreted proteins were found. This work presents the large scale of the rice secretory proteome from culture medium, which contributes to a deeper understanding of the rice secretome.     

Detailed proteome analysis of growing cells of the planctomycete Rhodopirellula baltica SH1T

Rhodopirellula baltica SH1(T), which was isolated from the water column of the Kieler Bight, a bay in the southwestern Baltic Sea, is a marine aerobic, heterotrophic representative of the ubiquitous bacterial phylum Planctomycetes. We analyzed the R. baltica proteome by applying different preanalytical protein as well as peptide separation techniques (1-D and 2-DE, HPLC separation) prior to MS. That way, we could identify a total of 1115 nonredundant proteins from the intracellular proteome and from different cell wall protein fractions. With the contribution of 709 novel proteins resulting from this study, the current comprehensive R. baltica proteomic dataset consists of 1267 unique proteins (accounting for 17.3% of the total putative protein-coding ORFs), including 261 proteins with a predicted signal peptide. The identified proteins were functionally categorized using Clusters of Orthologous Groups (COGs), and their potential cellular locations were predicted by bioinformatic tools. A unique protein family that contains several YTV domains and is rich in cysteine and proline was found to be a component of the R. baltica proteinaceous cell wall. Based on this comprehensive proteome analysis a global schema of the major metabolic pathways of growing R. baltica cells was deduced.     

A Preliminary Analysis of the Proteins of the Primary Walls of some Plant Cells

Amino-acid analyses on hydrolysates of the wall, microsomal,and soluble fractions of Jerusalem artichoke tubers, pea stems,and oat coleoptiles showed that the wall fractions alone containedhydroxyproline. In the case of Jerusalem artichoke, this substancewas positively identified as 1-hydroxyproline. In the electronmicroscope, contamination of the wall fraction by cytoplasmand cytoplasmic membrane was found to be negligible. Analyseson Jerusalem artichoke tuber tissue showed that the amountsof protein and hydroxyprolme in the walls increased with growth.It is concluded that a distinct group of proteins containinghydroxyproline form an integral part of the primary walls ofthese tissues.     

A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue

Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties.