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Fulfilling the promise: drug discovery in the post-genomic era.

The genomic era has brought with it a basic change in experimentation, enabling researchers to look more comprehensively at biological systems. The sequencing of the human genome coupled with advances in automation and parallelization technologies have afforded a fundamental transformation in the drug target discovery paradigm, towards systematic whole genome and proteome analyses. In conjunction with novel proteomic techniques, genome-wide annotation of function in cellular models is possible. Overlaying data derived from whole genome sequence, expression and functional analysis will facilitate the identification of causal genes in disease and significantly streamline the target validation process. Moreover, several parallel technological advances in small molecule screening have resulted in the development of expeditious and powerful platforms for elucidating inhibitors of protein or pathway function. Conversely, high-throughput and automated systems are currently being used to identify targets of orphan small molecules. The consolidation of these emerging functional genomics and drug discovery technologies promises to reap the fruits of the genomic revolution.

Animals↗

[Effect of some active Chinese herbal fraction on brain tissue proteomic profile of ischemic mouse].

OBJECTIVE: To study the effect of some active Chinese herbal fraction on protein expression of brain tissue in ischemic mouse with proteomic technique. METHODS: Ischemia-reperfusion mice were treated with baicalin, geniposide, cholic acid and concha margaritifera respectively for 3 hrs, and then their brain tissue were taken to extract the total protein. Protein expression in ischemic mouse brain was analyzed with surface-enhanced laser desorption/inionation-time of flight-mass spectra (SELDI-TOF-MS) protein-chip. RESULTS: The four components tested had effect on 3 target proteins at 5373Da, 5707Da and 15103Da, showing the nature of multi-target and with different action on protein expression. CONCLUSION: Protein-chip is an effective approach for exploring the pharmacological mechanism of Chinese herbal fraction.

Animals↗

A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells.

In the subventricular zone of the adult mammalian forebrain, neural stem cells (NSCs) reside and proliferate to generate young neurons. We screened factors that promoted the proliferation of NSCs in vitro by a recently developed proteomics technique, the ProteinChip system. In this screen, we identified a soluble carbohydrate-binding protein, Galectin-1, as a candidate. We show herein that Galectin-1 is expressed in a subset of slowly dividing subventricular zone astrocytes, which includes the NSCs. Based on results from intraventricular infusion experiments and phenotypic analyses of knockout mice, we demonstrate that Galectin-1 is an endogenous factor that promotes the proliferation of NSCs in the adult brain.

Animals↗

Characterization and location of post-translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules.

Bovine chromoganin B (CGB)/secretogranin I, an acidic protein with a sequence of 626 residues and an isoelectric point of 5.2 is a major member of the chromogranin/secretogranin (CG/Sg) family. The difference between the theoretical molecular mass (76 kDa) and the value estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis results from post-translational modifications (glycosylation, phosphorylation and sulfation) and from the abundance of acidic residues (D 4.6%, and E 16.5%). Although the sequence of CGB is known, the structural analyses of the post-translational modifications have so far not been carried out. In the present study, using a combination of proteomic techniques including two-dimensional gel electrophoresis, Western blot, high-performance liquid chromatography purification, enzymatic digestion, sequencing, carbohydrate analysis, matrix-assisted laser desorption/ionization-time of flight and liquid chromatography mass spectrometry analysis, we have located 18 post-translational modifications on bovine CGB, isolated from adrenal medulla chromaffin granules. Furthermore, we have identified at the molecular level the presence of a mutation M/V on position 577 of natural CGB. All together these data reflect the complex structure of this protein marker of the neuroendocrine system.

Adrenal Medulla↗

New Escherichia coli outer membrane proteins identified through prediction and experimental verification.

Many new Escherichia coli outer membrane proteins have recently been identified by proteomics techniques. However, poorly expressed proteins and proteins expressed only under certain conditions may escape detection when wild-type cells are grown under standard conditions. Here, we have taken a complementary approach where candidate outer membrane proteins have been identified by bioinformatics prediction, cloned and overexpressed, and finally localized by cell fractionation experiments. Out of eight predicted outer membrane proteins, we have confirmed the outer membrane localization for five-YftM, YaiO, YfaZ, CsgF, and YliI--and also provide preliminary data indicating that a sixth--YfaL--may be an outer membrane autotransporter.

Bacterial Outer Membrane Proteins↗

Proteomic identification of the wt-p53-regulated tumor cell secretome.

Tumor-stroma interactions play a major role in tumor development, maintenance and progression. Yet little is known on how the genetic alterations that underlie cell transformation elicit cell extrinsic changes modulating heterotypic cell interactions. We hypothesized that these events involve a modification in the complement of secreted proteins by the cell, acting as mediators of intercellular communication. To test this hypothesis, we examined the role of wt-p53, a major tumor suppressor, on the tumor microenvironment through its regulation of secreted factors. Using a combination of 2-DE and cICAT proteomic techniques, we found a total of 111 secreted proteins, 39 of which showed enhanced and 21 inhibited secretion in response to wt-p53 expression. The majority of these were not direct targets of p53 transcription factor activity, suggesting a novel role for wt-p53 in the control of intracellular protein trafficking and/or secreted protein stability. Evidence for p53-controlled post-translational modifications on nine secreted proteins was also found. These findings will enhance our understanding of wt-p53 modulated interactions of the tumor with its environment.

Cell Line, Tumor↗

Multi-Q: a fully automated tool for multiplexed protein quantitation.

The iTRAQ labeling method combined with shotgun proteomic techniques represents a new dimension in multiplexed quantitation for relative protein expression measurement in different cell states. To expedite the analysis of vast amounts of spectral data, we present a fully automated software package, called Multi-Q, for multiplexed iTRAQ-based quantitation in protein profiling. Multi-Q is designed as a generic platform that can accommodate various input data formats from search engines and mass spectrometer manufacturers. To calculate peptide ratios, the software automatically processes iTRAQ's signature peaks, including peak detection, background subtraction, isotope correction, and normalization to remove systematic errors. Furthermore, Multi-Q allows users to define their own data-filtering thresholds based on semiempirical values or statistical models so that the computed results of fold changes in peptide ratios are statistically significant. This feature facilitates the use of Multi-Q with various instrument types with different dynamic ranges, which is an important aspect of iTRAQ analysis. The performance of Multi-Q is evaluated with a mixture of 10 standard proteins and human Jurkat T cells. The results are consistent with expected protein ratios and thus demonstrate the high accuracy, full automation, and high-throughput capability of Multi-Q as a large-scale quantitation proteomics tool. These features allow rapid interpretation of output from large proteomic datasets without the need for manual validation. Executable Multi-Q files are available on Windows platform at http://ms.iis.sinica.edu.tw/Multi-Q/.

Chromatography, Liquid↗

Use of proteomic analysis of endometriosis to identify different protein expression in patients with endometriosis versus normal controls.

OBJECTIVE: To use proteomic techniques, including two-dimensional electrophoresis (2-DE), Western blot, and mass spectrometry, to screen and identify proteins that were expressed differently in patients with endometriosis versus normal controls. DESIGN: First, we aimed to find a difference in the way serum and eutopic endometrial proteins were expressed in women with and without endometriosis. Second, we were interested in searching for endometriotic proteins, which were specifically recognized by sera from patients with endometriosis. SETTING: Collaborative investigation in an academic research environment. PATIENT(S): Consenting women of reproductive age taking no medications and with laparoscopically proven endometriosis. INTERVENTION(S): Surgical excision of eutopic and ectopic endometrial biopsy and phlebotomization of patients with endometriosis and controls. MAIN OUTCOME MEASURE(S): Protein expression. RESULT(S): Thirteen protein spots from serum correlated with 11 known proteins and 11 protein spots from endometrium correlated with 11 known proteins were found differently expressed between women with and without endometriosis. Some proteins may be cytoskeletons, and some may be involved in the regulation of cell cycle, signal transduction, or immunological function. Three proteins, which were identified as vimentin, beta-actin, and ATP synthase beta subunit, hybridized significantly differently between endometriosis sera and normal sera. CONCLUSION(S): The data help to establish a human endometriosis proteome database and broaden our understanding of the pathogenesis of endometriosis. Further study of the proteins identified herein will assist in the eventual development of new diagnoses and treatments for endometriosis.

Actins↗

Analytical challenges for mapping non-canonical and non-protein ubiquitin/Ubl modifications by mass spectrometry.

INTRODUCTION: Covalent modification by ubiquitin via Lys isopeptide bonds is fundamental for regulating protein turnover and function. Additionally, ubiquitin esterification occurs on Ser/Thr/Tyr residues in proteins and on non-proteinaceous substrates including ribose, saccharides, lipids, and small molecule drugs. Ubiquitin posttranslational modifications may therefore be much more widespread across cell biological pathways. Recent literature (PubMed) reflects the increased interest in analytical methods for mapping of non-canonical substrates modified by ubiquitin and ubiquitin-like (UBL) proteins. AREAS COVERED: Mass spectrometry (MS)-based methodologies involve advanced proteomic techniques to identify ubiquitin modifications on amino acids other than Lys, such as Ser, Thr, Tyr and Cys as well as protein N-termini. After digestion, standard MS workflows identify canonical ubiquitination by detecting a ubiquitin C-terminal tag attached to the amine side chains of Lys residues of substrate-derived peptides suitable for MS/MS sequencing. For non-canonical modifications on proteins and substrates other than proteins, specialized strategies are required, such as using antibodies to enrich N-terminally modified peptides in combination with using high-resolution MS/MS based on softer fragmentation technologies to detect esterification and possibly other types of substrate modifications. EXPERT OPINION: Enabling such technologies will reveal a previously unrecognized angle of the ubiquitin code's complexity in cells.

Humans↗

A proteomics approach to identify changes in protein profiles in pre-cancerous colon.

The development of colon cancer is characterised by alterations in multiple genetic and epigenetic pathways in colon tissue leading ultimately to deregulation of colon epithelial cells. Early detection is an important factor in decreasing colon cancer deaths. Proteomic techniques were used to identify potential early markers in colon tissue exhibiting pre-cancerous activity that may characterise pathological changes in a chemically induced colon cancer rat model. Protein profiles were assessed in soluble and insoluble fractions prepared from distal colon of rats treated with the colonotropic carcinogen, dimethylhydrazine. Alterations in protein profiles were associated with the presence of aberrant crypt foci, hyperplasia and dysplasia, microanatomical changes, and metabolic changes in rat colon. These changes may have a potential role in the identification of pre-pathological features preceding colon tumorigenesis.

Animals↗

Comparative analysis of whole saliva proteomes for the screening of biomarkers for oral lichen planus.

OBJECTIVE: To screen possible biomarkers in whole saliva from oral lichen planus (OLP) patients by proteomic techniques. METHODS: Two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were applied to whole saliva of OLP patients and controls, and the differential protein components were analyzed with peptide mass fingerprinting and database searching. RESULTS: A total of 31 protein spots representing 14 proteins with at least two-fold difference in abundance between OLP and controls were identified. Among these, the expression of urinary prokallikrein was increased while short palate, lung and nasal epithelium carcinoma associated protein (PLUNC) was decreased in all samples of OLP. CONCLUSIONS: Urinary prokallikrein and PLUNC may be the new biomarkers which play a role in inflammation and immune response of OLP.

Adult↗

Proteomic study of cold shock protein in Bacillus stearothermophilus P1: Comparison of temperature downshifts.

The thermophilic bacterium Bacillus stearothermophilus P1 is unique in its ability to thrive in extreme environments such as high temperatures or high pH conditions. The study of cold shock response is very interesting and interpreted as a shock response to express the genes involved in synthesis of specific proteins. This study investigated the study of cold shock protein of B. stearothermophilus P1 when the cell culture temperature shifted from 65 degrees C to 37 degrees C and 25 degrees C. Cell growth at 37 degrees C weakly increased in the previous 3 h and then slowly decreased. In contrast, cell growth at 25 degrees C was slowly decreased. The protein contents after temperature downshifts were analyzed by proteomic techniques using protein chip and two-dimensional (2-D) electrophoresis that are highly effective and useful for protein separation and identification. The different proteins after a temperature decrease from 65 degrees C to 37 degrees C and 25 degrees C were expressed on 2-D gel patterns and the cold shock protein was detected in the acidic area with the isoelectric point and molecular mass approximately 4.5 and 7.3 kDa, respectively. The NH(2)-terminal sequence of a major cold shock protein from B. stearothermophilus P1 was MQRGKVKWFNNEKGFGFIEVEGGSD, similar to other cold shock proteins from Bacillus sp. up to 96% identity, but different from the other bacteria with homology less than 80% identity.

Bacterial Proteins↗

Halophile aldehyde dehydrogenase from Halobacterium salinarum.

Halobacterium salinarum is a member of the halophilic archaea. In the present study, H. salinarum was cultured at various NaCl concentrations (3.5, 4.3, and 6.0 M NaCl), and its proteome was determined and identificated via proteomics technique. We detected 14 proteins which were significantly down-regulated in 3.5 M and/or 6 M NaCl. Among the identified protein spots, aldehyde dehydrogenase (ALDH) was selected for evaluation with regard to its potential applications in industry. The most effective metabolism function exhibited by ALDH is the oxidation of aldehydes to carboxylic acids. The ALDH gene from H. salinarum (1.5 kb fragment) was amplified by PCR and cloned into the E. coli strain, BL21 (DE3), with the pGEX-KG vector. We subsequently analyzed the enzyme activity of the recombinant ALDH (54 kDa) at a variety of salt concentrations. The purified recombinant ALDH from H. salinarum exhibited the most pronounced activity at 1 M NaCl. Therefore, the ALDH from H.salinarum is a halophilic enzyme, and may prove useful for applications in hypersaline environments.

Aldehyde Dehydrogenase↗

MS1, MS2, and SQT-three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and identifications.

As the speed with which proteomic labs generate data increases along with the scale of projects they are undertaking, the resulting data storage and data processing problems will continue to challenge computational resources. This is especially true for shotgun proteomic techniques that can generate tens of thousands of spectra per instrument each day. One design factor leading to many of these problems is caused by storing spectra and the database identifications for a given spectrum as individual files. While these problems can be addressed by storing all of the spectra and search results in large relational databases, the infrastructure to implement such a strategy can be beyond the means of academic labs. We report here a series of unified text file formats for storing spectral data (MS1 and MS2) and search results (SQT) that are compact, easily parsed by both machine and humans, and yet flexible enough to be coupled with new algorithms and data-mining strategies.

Database Management Systems↗

Use of proteomic methodology for the characterization of human milk fat globular membrane proteins.

Characterization of the major human milk fat globular membrane proteins was carried out using proteomic techniques comprising two-dimensional polyacrylamide gel electrophoresis, followed by in situ PNGase F and trypsin digestion. Matrix-assisted laser desorption/ionization quadrupole time-of-flight and electrospray ionization mass spectrometry identified seven major protein components: alpha-lactalbumin, lysozyme precursor, beta-casein, clusterin, lactotransferrin, polymeric immunoglobulin receptor precursor, and human milk fat globule EGF-factor 8 protein. Sequence information on the protein-associated glycans was determined by matrix-assisted laser desorption-ionization quadrupole time-of-flight hybrid mass spectrometry. This glycan analysis revealed interesting fucosylation branching patterns which may be influential in maternal protection of the newborn against bacterial and viral pathogenic attack.

Amino Acid Sequence↗

Quantitative profiling of the membrane proteome in a halophilic archaeon.

We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotope-coded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small.

Cell Membrane↗

How does Pasteurella multocida respond to the host environment?

Pasteurella multocida is a Gram-negative bacterial pathogen, which causes diseases of economic importance in a wide range of animal species. The response of P. multocida to the host environment has been analysed at the transcription level, using DNA microarrays, and at the protein-expression level, using proteomics techniques. Furthermore, a growing number of P. multocida-directed mutants have been assessed for their ability to cause disease. Although technical impediments mean that it is currently difficult to analyse bacterial responses at the earliest stages of infection, it is clear that during later stages of infection the bacteria encounter host niches that require them to modify the expression of genes involved in central energy metabolism and in the uptake of various nutrients such as iron and amino acids. Furthermore, in vitro experiments have defined the varying bacterial responses to low iron and to different iron sources, including haemoglobin and transferrin. To date, most P. multocida genes shown to be upregulated during infection are involved in nutrient acquisition and metabolic processes, indicating that true virulence genes might be constitutively expressed, upregulated only during initial stages of infection or upregulated at levels below current detection limits.

Adaptation, Physiological↗

Subunit architecture of multimeric complexes isolated directly from cells.

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.

Chromatography, Affinity↗