Prefractionation by digitonin extraction increases representation of the cytosolic and intracellular proteome of Leishmania infantum

Proteome coverage is limited by the dynamic range of proteins present in a sample and often is confined to the analysis of abundant proteins. We have developed a protein prefractionation protocol, based on the differential solubilization of membranes using digitonin, that has allowed an increase in the resolution and depth of comparative proteomic studies. This prefractionation protocol can also be used to infer the subcellular localization of hypothetical proteins as tested experimentally using green fluorescent fusion proteins. The abundant tubulins and associated proteins of the cytoskeleton were removed from the sample using digitonin extraction, hence facilitating the visualization of lower abundance proteins. The digitonin prefractionation protocol was applied for a comparative proteomic analysis of the promastigote and amastigote life cycle stages of Leishmania infantum and has allowed the identification of novel proteins expressed in a stage-specific manner.     

15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.     

Crystallographic studies on B12 binding proteins in eukaryotes and prokaryotes

The X-ray crystal structures of several important vitamin B12 binding proteins that have been solved in recent years have enhanced our current understanding in the vitamin B12 field. These structurally diverse groups of B12 binding proteins perform various important biological activities, both by transporting B12 as well as catalyzing various biological reactions. An in-depth comparative analysis of these structures was carried out using PDB coordinates of a carefully chosen database of B12 binding proteins to correlate the overall folding of the molecule with phylogeny, the B12 interactions, and with their biological function. The structures of these proteins are discussed in the context of this comparative analysis.     

Prediction of functionally related proteins by comparative genomics in silico

The review considers the computational prediction of functionally related proteins by comparative genomics. Growing possibilities of biotechnology for genome sequencing lead to generation of sequences for millions of genes. However, functions of majority of these genes remain unknown, and can be determined experimentally only for a few of them. Therefore, accurate and robust methods for in silico prediction (annotation) of gene functions are needed. We describe here the main techniques of comparative genomics, including the standard method based on transferring functions between homologous sequences and also context-based methods, including phylogenetic profiles and gene-neighbor approaches. Modern methods of comparative genomics allow obtaining correct functional annotations for more than a half of all organism proteins.     

Comparison of algorithms for prediction of related proteins using the method of phylogenetic profiles

Computational interactomics deals with prediction of functionally related proteins. One approach for solving this problem using comparative genomics consists in analysis of similarities between phylogenetic profiles of proteins. In contrast to most methods, which predict only pairwise interactions between proteins, in the present work we have applied the cluster analysis techniques in order to find modules of functionally related proteins. We have performed the cluster analysis of phylogenetic profiles of E. coli proteins using several clustering techniques and various modes for estimation of distances between profiles. We report here, that the best correspondence in the composition of resultant clusters to known metabolic pathways is achieved using Ward’s clustering together with Hamming’s distance. The proposed technique of assessing predictions of the modules of functionally related proteins can be used for comparative analysis of different algorithms for computational interactomics.     

Distance-dependent statistical potentials for discriminating thermophilic and mesophilic proteins

Identification of the characteristic structural patterns responsible for protein thermostability is theoretically important and practically useful but largely remains an open problem. These patterns may be revealed through comparative study on thermophilic and mesophilic proteins that have distinct thermostability. In this study, we constructed several distance-dependant potentials from thermophilic and mesophilic proteins. These potentials were then used to evaluate the structural difference between thermophilic and mesophilic proteins. We found that using the subtraction or division of the potentials derived from thermophilic and mesophilic proteins can dramatically increase the discriminatory ability. This approach revealed that the ability to distinct the subtle structural features responsible for protein thermostability may be effectively enhanced through rationally designed comparative study.     

Who's your neighbor? New computational approaches for functional genomics

Several recently developed computational approaches in comparative genomics go beyond sequence comparison. By analyzing phylogenetic profiles of protein families, domain fusions, gene adjacency in genomes, and expression patterns, these methods predict many functional interactions between proteins and help deduce specific functions for numerous proteins. Although some of the resultant predictions may not be highly specific, these developments herald a new era in genomics in which the benefits of comparative analysis of the rapidly growing collection of complete genomes will become increasingly obvious.     

Comparative proteomic analysis of peripheral blood mononuclear cells from atopic dermatitis patients and healthy donors

Atopic dermatitis (AD) is a chronic inflammatory skin disease that induces changes in various inflammatory skin cells. The prevalence of AD is as high as 18% in some regions of the world, and is steadily rising. However, the pathophysiology of AD is poorly understood. To identify the proteins involved in AD pathogenesis, a comparative proteomic analysis of protein expression in peripheral blood mononuclear cells isolated from AD patients and healthy donors was conducted. Significant changes were observed in the expressions of fourteen proteins, including the vinculin, PITPNB, and Filamin A proteins. Among the proteins, alpha-SNAP and FLNA decreased significantly, and PITPNB increased significantly in AD patients compared with control subjects; these findings were further confirmed by real-time PCR and Western blot analysis. The comparative proteome data may provide a valuable clue to further understand AD pathogenesis, and several differentially regulated proteins may be used as biomarkers for diagnosis and as target proteins for the development of novel drugs.     

Investigation of doxorubicin resistance in MCF-7 breast cancer cells using shot-gun comparative proteomics with proteolytic 18O labeling

A shot-gun comparative proteomic investigation utilizing proteolytic 18O labeling has been carried out on a drug susceptible MCF-7 human breast cancer cell line and a related cell line that is resistant to doxorubicin. The proteolytic 18O labeling method has been further refined and optimized for application to a protein fraction stemming from the cytosol of the breast cancer cells. The comparative investigation revealed several proteins with altered expression levels in the doxorubicin resistant line. These altered proteins are considered for a possible role in doxorubicin resistance.     

High-resolution two-dimensional electrophoresis of nuclear proteins: a comparison of HeLa nuclei prepared by three different methods.

A comparative analysis of HeLa cell nuclear proteins is presented using Iso-Dalt methods of protein resolution in two dimensions. The nuclear proteins were prepared by (1) spin through glycerol cushion, (2) spin through sucrose cushion, or (3) Triton wash. Improved resolution of total nuclear proteins in the range of pH 4.5-6.0 was achieved by substituting longer isotubes in combination with broad-range ampholines during the isoelectric focusing step. An attempt to indicate silver stainable protein spots common to total cellular extracts and nuclear preparations has been made. Also, proteins that appear to be well represented in all three nuclear preparations and remain undetectable in the total cellular protein pattern have been marked as probably being enriched nuclear proteins. Such a comparative analysis of whole nuclear protein preparations made it possible to document that the different preparations preserved the same set of proteins. The Triton-wash method of obtaining nuclei was identified as the preferred choice. Coomassie-stained gels and blots of these nuclear proteins could serve as a guide for accessing relevant protein spots for further biochemical analysis such as immunoblotting.     

Identification of proteins with altered expression in colorectal cancer by means of 2D-proteomics

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumors as compared to normal tissues. Although proteomics is currently widely used, identification of proteins differentially expressed in particular types of cancer remains a challenging task. The goal of our study was to detect novel protein markers of colorectal cancer using comparative proteomics of protein extracts obtained from primary tumors and adjacent normal tissues. Coloreetal cancer is nearly asymptomatic at the early stages, which calls for development of fast and sensitive methods for molecular diagnostics. Proteomes of 11 paired specimens of primary colorectal tumors and adjacent histologically normal tissues were studied using comparative 2D PAGE. Altogether, 16 proteins with altered expression levels were detected, including 13 proteins with increased levels and three proteins with decreased levels in tumor tissues. These proteins were identified using MALDI-TOF mass spectrometry. The proteins GPD1, RRBP1 (increased levels), HNRNPH1, and SERPINB6 (decreased levels) have been associated with colorectal cancer for the first time.     

TASSER-Lite: an automated tool for protein comparative modeling

This study involves the development of a rapid comparative modeling tool for homologous sequences by extension of the TASSER methodology, developed for tertiary structure prediction. This comparative modeling procedure was validated on a representative benchmark set of proteins in the Protein Data Bank composed of 901 single domain proteins (41-200 residues) having sequence identities between 35-90% with respect to the template. Using a Monte Carlo search scheme with the length of runs optimized for weakly/nonhomologous proteins, TASSER often provides appreciable improvement in structure quality over the initial template. However, on average, this requires approximately 29 h of CPU time per sequence. Since homologous proteins are unlikely to require the extent of conformational search as weakly/nonhomologous proteins, TASSER's parameters were optimized to reduce the required CPU time to approximately 17 min, while retaining TASSER's ability to improve structure quality. Using this optimized TASSER (TASSER-Lite), we find an average improvement in the aligned region of approximately 10% in root mean-square deviation from native over the initial template. Comparison of TASSER-Lite with the widely used comparative modeling tool MODELLER showed that TASSER-Lite yields final models that are closer to the native. TASSER-Lite is provided on the web at (http://cssb.biology.gatech.edu/skolnick/webservice/tasserlite/index.html).     

Geometrical parameters and shape anisotropy in globular proteins

The anisotropy in the shape of globular proteins is derived by a comparative analysis of three types of geometrical parameters. The role of secondary structures in the design of the shape of globular proteins is worked out.     

Comparative analysis of cell surface proteins in chronic and acute leukemia cell lines

This study was designed to identify the cell surface protein markers that can differentiate between chronic myeloid leukemia (CML) and acute promyelocytic leukemia cells (APL). The differentially expressed plasma membrane proteins were analyzed between CML cell line (K562) and APL cell line (NB4) using the comparative proteomic approach. The cell membrane proteins were enriched by labeling with a membrane-impermeable biotinylation reagent, sulfo-NHS-SS-Biotin, and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). By comparative proteomic analysis of K562 and NB4 cells, we identified 25 membrane and 14 membrane-associated proteins. The result of LC-MS/MS combined with chemical tagging method was validated by confirming the expression and localization of one of the differentially expressed plasma membrane proteins, CD43, by FACS and confocal microscopy. Our results indicate that CD43 could be a potential candidate for differentiating CML from APL.     

Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five noncysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.     

Was the evolution of plastid genetic machinery discontinuous?

The plastid nucleoid consists of plastid DNA and various, mostly uncharacterized, DNA-binding proteins. The plastid DNA undoubtedly originated from an ancestral cyanobacterial genome, but the origin of the nucleoid proteins appears complex. Initial biochemical analysis of these proteins, as well as comparative genome informatics, suggest that proteins of eukaryotic origin replaced most of the original prokaryotic proteins during the evolution of plastids in the lineage of green plants.     

Proteomic characterization of the greening process in rice seedlings using the MS spectral intensity-based label free method

Illumination-induced greening in dark-grown plants is one of the most dramatic developmental processes known in plants. In our current study, we characterized the greening process of rice seedlings using comparative proteome analysis. We identified 886 different proteins in both whole cell lysates of illuminated and nonilluminated rice shoots and performed comparative proteome analysis based on the MS spectral intensities obtained for unique peptides from respective proteins. Furthermore, the changes in the levels of individual proteins were then compared with those of the corresponding mRNAs. The results revealed well-coordinated increases in the enzymes involved in the Calvin cycle at both the protein and mRNA levels during greening, and that the changes at the mRNA level precede those at the protein level. Although a much lower effect of illumination was found on the enzymes associated with glycolysis and the TCA cycle, coordinated increases during greening were evident for the enzymes involved in photorespiration and nitrogen assimilation as well as the components of the chloroplastic translational machinery. These results thus define the differential regulation of distinct biological systems during greening in rice and demonstrate the usefulness of comprehensive and comparative proteome analysis for the characterization of biological processes in plant cells.     

Targeted tissue proteomic analysis of human astrocytomas

Complicating proteomic analysis of whole tissues is the obvious problem of cell heterogeneity in tissues, which often results in misleading or confusing molecular findings. Thus, the coupling of tissue microdissection for tumor cell enrichment with capillary isotachophoresis-based selective analyte concentration not only serves as a synergistic strategy to characterize low abundance proteins, but it can also be employed to conduct comparative proteomic studies of human astrocytomas. A set of fresh frozen brain biopsies were selectively microdissected to provide an enriched, high quality, and reproducible sample of tumor cells. Despite sharing many common proteins, there are significant differences in the protein expression level among different grades of astrocytomas. A large number of proteins, such as plasma membrane proteins EGFR and Erbb2, are up-regulated in glioblastoma. Besides facilitating the prioritization of follow-on biomarker selection and validation, comparative proteomics involving measurements in changes of pathways are expected to reveal the molecular relationships among different pathological grades of gliomas and potential molecular mechanisms that drive gliomagenesis.     

Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis

The ability to identify and quantitate integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and facilitate derivatization of these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified, of which 40 were integral membrane proteins containing from one to nine mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g., ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g., pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.