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PubMed2002

Controlling deuterium isotope effects in comparative proteomics.

This paper focuses on identifying structural features responsible for resolution of heavy isotope coded peptides during reversed-phase chromatography. This was achieved by using labeled coding agents that varied in structure, number of deuterium atoms, placement of deuterium in the coding agent, and the functional group targeted by the reagent. Six coding agents were examined. Deuterated versions of the coding agents studied included succinic anhydride-2H4, acetic acid 2,5-dioxopyrrolidin-1-yl ester-2H3, propionic acid 2,5-dioxopyrrolidin-1-yl ester-2H5, pentanoic acid 2,5-dioxopyrrolidin-1-yl ester-2H9, [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)-propyl]-trimethylammonium chloride-2H9, and the commercial ICAT-2H8 reagent. It was found that these labeling agents vary widely in both their absolute and relative contribution to the chromatographic isotope effect. Relative effects were evaluated by normalizing resolution for the number of deuterium atoms in the derivatized peptide. The single, most dominant effect was the placement of deuterium atoms relative to hydrophilic functional groups in the coding agent. It was concluded that the probability of a deuterium atom interacting with the stationary phase of a reversed-phase chromatography (RPC) column and impacting resolution is greatly diminished by placing it adjacent to a hydrophilic group, as explained by solvophobic theory. But peptide size and coding agent size were also seen to correlate inversely with the magnitude of the isotope effect. This effect was explained as being due to the relative size of the coding agent versus that of the coding agent-peptide conjugate.

Animals↗
PubMed2005

Targeted comparative proteomics by liquid chromatography-tandem Fourier ion cyclotron resonance mass spectrometry.

In proteomics, effective methods are needed for identifying the relatively limited subset of proteins displaying significant changes in abundance between two samples. One way to accomplish this task is to target for identification by MS/MS only the "interesting" proteins based on the abundance ratio of isotopically labeled pairs of peptides. We have developed the software and hardware tools for online LC-FTICR MS/MS studies in which a set of initially unidentified peptides from a proteome analysis can be selected for identification based on their distinctive changes in abundance following a "perturbation". We report here the validation of this method using a mixture of standard proteins combined in different ratios after isotopic labeling. We also demonstrate the application of this method to the identification of Shewanella oneidensis peptides/proteins exhibiting differential abundance in suboxic versus aerobic cell cultures.

Bacterial Proteins↗
PubMedDate not supplied

Minimizing resolution of isotopically coded peptides in comparative proteomics.

Stable isotopes are now widely used to quantify concentration changes in proteomics. This paper focuses on the resolution of isotopically coded peptides and how isotope effects occurring during chromatographic separations can be minimized. Heavy isotope derivatizing agents used in this work were the commercially available 2H8-ICAT reagent and 13C4-succinic anhydride. The ICAT reagent derivatizes cysteine-containing peptides, whereas the succinic anhydride reacts with primary amine groups in peptides. It was observed during reversed-phase chromatography of peptides from a BSA tryptic digest differentially labeled with the 2Hr and 2H8-ICAT reagents that resolution of the isoforms exceeded 0.5 with 20% of the peptides in the digest. Three-fourths of the peptides in this group contained two cysteine residues and were doubly labeled. Only 23% of the peptides labeled with a single ICAT residue had a resolution greater than 0.4. The resolution of peptides differentially labeled with 13C- and 12C-succinate never exceeded +/- 0.01, even in the case of peptides from the BSA digest labeled with 2 mol of succinate. Because this value is within the limits of the method used to determine resolution, it was concluded the 13C- and 12C-coded isoforms of labeled peptides did not resolve. The isotope ratio in the case of 13C/12C coding could be determined from a single mass spectrum taken at any point in the elution profile. This enabled isotope ratio analysis to be completed early in the elution of a peptide from chromatography columns.

Carbon Isotopes↗
PubMedDate not supplied

Quantitation using enhanced signal tags: a technique for comparative proteomics.

Differential amidination of N-termini and lysine residues provides the basis for a novel approach to protein quantitation using MALDI mass spectrometry. Because the amidination of lysines increases their basicity and therefore MALDI ionization yields, the method is called quantitation using enhanced signal tags (QUEST). Amidine labels differ by methylene groups, leading to 14 Da mass differentials. The utility of QUEST is demonstrated while analyzing the digests of two model proteins using MALDI-TOF mass spectrometry.

Amidines↗
PubMedDate not supplied

Metabolic labeling of human primary retinal pigment epithelial cells for accurate comparative proteomics.

Metabolic labeling was evaluated, using both 13C6-Arg and 13C6, 15N2-Lys amino acids, for a primary human retinal pigment epithelial cell (hRPE) culture prepared from an autopsy eye of an 81 year old donor. Satisfactory incorporation (>90%) was achieved with both stable isotope labeled amino acids after four passages (roughly 7 population doublings). The degree of incorporation was found to be efficient with both amino acids as well as in different proteins. The presence of 10% whole serum in the culture medium did not interfere with the incorporation of the exogenous stable isotope labeled amino acids. Metabolic labeling of these human primary retinal pigment epithelial cells was further tested to quantify protein ratios between proliferating and resting cells using a combination of 2-DG and MALDI-TOF-TOF/MS analysis. Using computational data processing and analysis, we obtained accurate protein ratio measurement for every single identified protein (156 proteins) in the 2-Dg array. Of these 156 proteins, 12 proteins were found significantly increased in dividing versus resting cells by at least a factor of 1.5 while 13 other proteins were found increased in resting versus dividing cells by at least the same fold. Most of these differentially expressed proteins are directly involved in cell proliferation, protein synthesis, and actin-remodeling and differentiation.

Aged↗
PubMed2007

Comparative proteome analysis of serum from acute pulmonary embolism rat model for biomarker discovery.

Pulmonary embolism (PE) is a common, potentially fatal disease and its diagnosis is challenging because clinical signs and symptoms are nonspecific. In this study, to investigate protein alterations of a rat PE model, total serum proteins collected at different time points were separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Bioinformatics analysis of 24 differentially expressed proteins showed that 20 had corresponding protein candidates in the database. According to their properties and obvious alterations after PE, changes of serum concentrations of Hp, Fn, DBP, RBP, and TTR were selected to be reidentified by western blot analysis. Semiquantitative RT-PCR showed DBP, RBP, and TTR to be down-regulated at mRNA levels in livers but not in lung tissues. The low serum concentrations of DBP, RBP, and TTR resulted in the up-regulation of 25(OH)D3, vitamin A, and FT4 (ligands of DBP, RBP, and TTR) after acute PE in rat models. The serum levels of Hp and Fn were detected in patients with DVT/PE and controls to explore their diagnostic prospects in acute PE because the mRNA levels of Hp and Fn were found to be up-regulated both in lung tissues and in livers after acute PE. Our data suggested that the concentration of serum Fn in controls was 79.42 +/- 31.57 microg/L, whereas that of PE/DVT patients was 554.43 +/- 136.18 microg/L (P < 0.001), and that the concentration of serum Hp in controls was 824.37 +/- 235.24 mg/L, whereas that of PE/DVT patients was 2063.48 +/- 425.38 mg/L (P < 0.001). The experimental PE rat model selected in this study was more similar to the clinical process than the other existing PE animal models, and the findings indicated instant changes of serum proteins within 48 h after acute PE. The exploration of these differentially expressed proteins or their combination with existent markers such as D-dimer may greatly improve the accuracy of the diagnosis of acute PE, but diagnostic tests are still needed to evaluate the sensitivity and specificity of these markers and also the number of false positives and false negatives.

Acute Disease↗
PubMed2000

Comparative proteome analysis of Helicobacter pylori.

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.

Bacterial Proteins↗
PubMed2006

Comparative proteomics analysis of the proteins associated with laryngeal carcinoma-related gene 1.

OBJECTIVES: A novel gene, laryngeal carcinoma-related gene 1 (LCRG1), had the characteristics of tumor-suppressor genes. It was cloned in our laboratory. The objective was to find and characterize the proteins related to LCRG1 and to elucidate the molecular mechanism of LCRG1. STUDY DESIGN: We used the established cell lines of Hep-2/LCRG1 (Hep-2 cells transfected by recombinant plasmid pcDNA3.1[+]/LCRG1) and Hep-2/pcDNA3.1(+) (Hep-2 cells transfected by control vector pcDNA3.1[+]) as cell models. METHODS: Two-dimensional gel electrophoresis (2-DE) technology was performed to separate the proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines, respectively. The differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization-quadruple time-of-flight MS/MS (ESI-Q-TOF MS/MS). Then the differential expression levels of partial identified proteins were determined by Western blotting analysis and quantitative real-time reverse transcriptase-polymerase chain reaction. RESULTS: The results showed the attained 2-DE patterns of the two cell lines were well-resolved and reproducible. There were 1075+/-43 and 1027+/-23 protein spots observed in Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines, respectively. The average matching rate of the two cell lines was 91%. Twenty-six differentially expressed protein spots were identified (twenty spots for MALDI-TOF-MS, six spots for ESI-Q-TOF MS/MS). Most of the characterized proteins were characterized as the members of enzymes (phosphoglycerate mutase, manganese superoxide dismutase, and so on), transcription proteins (rho gdp dissociation inhibitor), and so on. Those identified proteins might contribute to the tumor-suppressive function of LCRG1. The differential expression levels of the partial proteins were confirmed by real-time polymerase chain reaction and Western blotting. CONCLUSIONS: We tentatively proposed those differentially expressed proteins were involved in the tumor-suppressive process related to LCRG1. These data will be helpful to elucidate the molecular mechanism of LCRG1.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Comparative proteomic analysis of metabolically labelled proteins from Plasmodium falciparum isolates with different adhesion properties.

The virulence of Plasmodium falciparum relates in part to the cytoadhesion characteristics of parasitized erythrocytes but the molecular basis of the different qualitative and quantitative binding phenotypes is incompletely understood. This paucity of information is due partly to the difficulty in working with membrane proteins, the variant nature of these surface antigens and their relatively low abundance. To address this two-dimensional (2D) protein profiles of closely related, but phenotypically different laboratory strains of P. falciparum have been characterized using proteomic approaches. Since the mature erythrocyte has no nucleus and no protein synthesis capability, metabolic labelling of proteins was used to selectively identify parasite proteins and increase detection sensitivity.A small number of changes (less than 10) were observed between four different P. falciparum laboratory strains with distinctive cytoadherence properties using metabolic labelling, with more parasite protein changes found in trophozoite iRBCs than ring stage. The combination of metabolic labelling and autoradiography can therefore be used to identify parasite protein differences, including quantitative ones, and in some cases to obtain protein identifications by mass spectrometry. The results support the suggestion that the membrane protein profile may be related to cytoadherent properties of the iRBCs. Most changes between parasite variants were differences in iso-electric point indicating differential protein modification rather than the presence or absence of a specific peptide.

Animals↗
PubMed2005

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes.

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.

Journal Article↗
PubMed2004

Comparative proteomic analysis of human CD34+ stem/progenitor cells and mature CD15+ myeloid cells.

Human CD34(+) cells, highly enriched for hematopoietic stem and progenitors, and CD15(+) cells, more terminally differentiated myeloid cells in blood, represent distinct maturation/differentiation stages. A proteomic approach was used to identify proteins differentially present in these two populations from human cord blood. Cytosolic proteins were extracted and subjected to two-dimensional gel electrophoresis followed by mass spectrometry. On average, 460 protein spots on each gel were detected; 112 and 15 proteins, respectively, were found to be differentially expressed or post-translationally modified in CD34(+) and CD15(+) cells. This suggests that CD34(+) cells have a relatively larger proteome than mature CD15(+) myeloid cells and production of many stem/progenitor cell-associated proteins ceases or is dramatically down-regulated as the CD34(+) cells undergo differentiation. Of approximately 140 protein spots, 47 different proteins were positively identified by mass spectrometry and database search; these proteins belong to several functional categories, including cell signaling, transcription factors, cytoskeletal proteins, metabolism, protein folding, and vesicle trafficking. Multiple heat shock proteins and chaperones, as well as proteins important for intracellular trafficking, were predominantly present in CD34(+) cells. Most of the identified proteins in CD34(+) cells are expressed in germ cell tumors, as well as in embryonal carcinoma and neuroblastoma. Approximately eight novel proteins, whose functions are unknown, were identified. This study presents, for the first time, global cellular protein expression patterns in human CD34(+) and CD15(+) cells, which should help to better understand intracellular processes involved in myeloid differentiation and add insight into the functional capabilities of these distinct cell types.

Antigens, CD34↗
PubMed2006

Tale of two metal reducers: comparative proteome analysis of Geobacter sulferreducens PCA and Shewanella oneidensis MR-1.

Geobacter sulfurreducens and S. oneidensis are the subjects of intense research efforts due to their potential applications to bioremediation. The characterization of their proteomes, being done in parallel with the analysis of their genome sequences, transcriptomes, and metabolomes, is providing valuable insights to both their similarities and their differences. A primary target of interest in the proteomes of both of these metal-reducing microbes is the characterization of their c-type cytochromes. The discovery of their full compliment of c-type cytochromes and the description of what growth conditions trigger their expression is central to harnessing their bioremediation potential. Proteome analyses thus far show that both G. sulfurreducens and S. oneidensis share the common location of a majority of their c-type cytochromes in their outer membranes. The c-type cytochromes of G. sulfurreducens, however, appear to be less soluble and therefore more difficult to isolate from the membranes than those expressed by S. oneidensis. The majority of the G. sulfurreducens c-type cytochromes also differ from those of S. oneidensis in that they have higher isoelectric points, most higher than pH 8.0. These characteristics of solubility and isoelectric point could be related and could indicate an underlying functional difference in the strategy for metal reduction between these two microbes. The global proteome results available for G. sulfurreducens and S. oneidensis at the time of this writing are primarily the result of 2DE analysis coupled to protein identification by LC-MS/MS of tryptic peptides from in-gel digests and represent the most abundant proteins detected by Coomassie blue or silver nitrate staining. Currently, several complimentary efforts utilising the 2D-LC-MS/MS approaches are in progress, promising a more complete protein inventory for these microbes in the near future. As these data are added to those already available, the intricate network of metabolic processes, regulation of protein synthesis and protein function, transport of nutrients, and signal transduction will be elucidated. The existing tools of proteomics will be complimented with newer methods such as protein chips and phage display to further characterize these microbial systems. The end result, in the not too distant future, will be predictive models of G. sulfurreducens and S. oneidensis behavior in their natural habitats under a variety of environmental conditions.

Bacterial Proteins↗
PubMed2024

Single-tissue proteomics in Caenorhabditis elegans reveals proteins resident in intestinal lysosome-related organelles.

The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry (MS)-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single-tissue MS-based proteomic analysis in small organisms and in its future utility.

Animals↗
PubMed2004

Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics.

Comparing different proteomes by classical two-dimensional electrophoresis is challenging and often complicated by substantial gel-to-gel variation. Separating two or more protein samples labelled with different fluorescent dyes in one single gel, as in two-dimensional difference gel electrophoresis, reduces this variability considerably. Recent technological innovations, specifically the introduction of a pooled internal standard, even further improve the quantification accuracy and statistical confidence of this method. In addition, decreasing the sample complexity by one of several protein or organelle fractionation procedures increases the number of spots investigated by this protein differential display methodology.

Animals↗
PubMed2006

The proteome of Mannheimia succiniciproducens, a capnophilic rumen bacterium.

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is an industrially important bacterium as an efficient succinic acid producer. Recently, its full genome sequence was determined. In the present study, we analyzed the M. succiniciproducens proteome based on the genome information using 2-DE and MS. We established proteome reference map of M. succiniciproducens by analyzing whole cellular proteins, membrane proteins, and secreted proteins. More than 200 proteins were identified and characterized by MS/MS supported by various bioinformatic tools. The presence of proteins previously annotated as hypothetical proteins or proteins having putative functions were also confirmed. Based on the proteome reference map, cells in the different growth phases were analyzed at the proteome level. Comparative proteome profiling revealed valuable information to understand physiological changes during growth, and subsequently suggested target genes to be manipulated for the strain improvement.

Animals↗
PubMed2001

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.

Animals↗
PubMed2005

Comparative genomics on Shisa orthologs.

WNT signaling molecules are implicated in a variety of human tumors, such as gastric cancer and colorectal cancer. FGFR2 gene, encoding FGF receptor 2, is amplified in human gastric and breast cancer. WNT and FGF signaling pathways network together during carcinogenesis and embryogenesis. Xenopus shisa is claimed to inhibit the post-translational maturation of wnt and fgf receptors. Here, we identified and characterized the rat Shisa (Tmem46) gene by using bioinformatics for comparative proteomics and comparative genomics analyses. Rat Shisa gene, consisting of two exons, was located within AC126002.4 genome sequence. Shisa gene at rat chromosome 15p12 was found to encode a type I transmembrane protein (295 aa), showing 99.3, 92.5, 81.7, 40.3 and 38.6% total-amino-acid identity with mouse Shisa, human SHISA, chicken shisa, Xenopus shisa and zebrafish shisa, respectively. The extracellular Cys-rich domain with eight Cys residues was conserved among vertebrate Shisa orthologs. The C-terminal cytoplasmic region was conserved among mammalian and chicken Shisa orthologs, but not in Xenopus and zebrafish Shisa orthologs. Human SHISA promoter and rat Shisa promoter were not conserved well. Function of human SHISA is predicted to be divergent from that of Xenopus shisa due to the protein evolution and the promoter evolution. This is the first report on the rat Shisa gene and on molecular evolution of Shisa orthologs.

Amino Acid Sequence↗