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PubMed2000

Proteomics: applications in basic and applied biology.

The rapid evolution of proteomics has continued during the past year, with a series of innovations in the core technologies of two-dimensional electrophoresis and mass spectrometry, and a diversity of productive research programmes. Well-annotated proteomics databases are now emerging in a number of fields to provide a platform for systematic research, with particularly promising progress in clinical applications such as cardiology and oncology. Large-scale quantitative research, comparable in power and sensitivity to that achieved for gene expression, is thus becoming a reality at the protein level.

Databases, Factual↗
PubMed2001

A neural network classifier capable of recognizing the patterns of all major subcellular structures in fluorescence microscope images of HeLa cells.

MOTIVATION: Assessment of protein subcellular location is crucial to proteomics efforts since localization information provides a context for a protein's sequence, structure, and function. The work described below is the first to address the subcellular localization of proteins in a quantitative, comprehensive manner. RESULTS: Images for ten different subcellular patterns (including all major organelles) were collected using fluorescence microscopy. The patterns were described using a variety of numeric features, including Zernike moments, Haralick texture features, and a set of new features developed specifically for this purpose. To test the usefulness of these features, they were used to train a neural network classifier. The classifier was able to correctly recognize an average of 83% of previously unseen cells showing one of the ten patterns. The same classifier was then used to recognize previously unseen sets of homogeneously prepared cells with 98% accuracy. AVAILABILITY: Algorithms were implemented using the commercial products Matlab, S-Plus, and SAS, as well as some functions written in C. The scripts and source code generated for this work are available at http://murphylab.web.cmu.edu/software. CONTACT: murphy@cmu.edu

Algorithms↗
PubMed2006

Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis.

To investigate aberrant plasma proteins in lung cancer, we compared the proteomic profiles of serum from five lung cancer patients and from four healthy volunteers. Immuno-affinity chromatography was used to deplete highly abundant plasma proteins, and the resulting plasma samples were separated into eight fractions by anion-exchange chromatography. Quantitative protein profiles of the fractionated samples were generated by two-dimensional difference gel electrophoresis, in which the experimental samples and the internal control samples were labeled with different dyes and co-separated by two-dimensional polyacrylamide gel electrophoresis. This approach succeeded in resolving 3890 protein spots. For 364 of the protein spots, the expression level in lung cancer was more than twofold different from that in the healthy volunteers. These differences were statistically significant (Student's t-test, p-value less than 0.05). Mass spectrometric protein identification revealed that the 364 protein spots corresponded to 58 gene products, including the classical plasma proteins and the tissue-leakage proteins catalase, clusterin, ficolin, gelsolin, lumican, tetranectin, triosephosphate isomerase and vitronectin. The combination of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis provides a valuable tool for serum proteomics in lung cancer.

Blotting, Western↗
PubMed2005

A proteomics study of in vitro cyst germination and appressoria formation in Phytophthora infestans.

A proteomics study using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed on Phytophthora infestans. Proteins from cysts, germinated cysts and appressoria grown in vitro were isolated and separated by 2-DE. Statistical quantitative analysis of the protein spots from five independent experiments of each developmental stage revealed significant up-regulation of ten spots on gels from germinated cysts compared to cysts. Five spots were significantly up-regulated on gels from appressoria compared to germinated cysts and one of these up-regulated spots was not detectable on gels from cysts. In addition, one spot was significantly down-regulated and another spot not detectable on the gels from appressoria. The corresponding proteins to 13 of these spots were identified with high confidence using tandem mass spectrometry and database searches. The functions of the proteins that were up-regulated in germinated cysts and appressoria can be grouped into the following categories: protein synthesis (e.g. a DEAD box RNA helicase), amino acid metabolism, energy metabolism and reactive oxygen species scavenging. The spot not detected in appressoria was identified as the P. infestans crinkling- and necrosis-inducing protein CRN2. The identified proteins are most likely involved in the establishment of the infection of the host plant.

Algal Proteins↗
PubMed2005

Comparison of tuber proteomes of potato varieties, landraces, and genetically modified lines.

Crop improvement by genetic modification remains controversial, one of the major issues being the potential for unintended effects. Comparative safety assessment includes targeted analysis of key nutrients and antinutritional factors, but broader scale-profiling or "omics" methods could increase the chances of detecting unintended effects. Comparative assessment should consider the extent of natural variation and not simply compare genetically modified (GM) lines and parental controls. In this study, potato (Solanum tuberosum) proteome diversity has been assessed using a range of diverse non-GM germplasm. In addition, a selection of GM potato lines was compared to assess the potential for unintended differences in protein profiles. Clear qualitative and quantitative differences were found in the protein patterns of the varieties and landraces examined, with 1,077 of 1,111 protein spots analyzed showing statistically significant differences. The diploid species Solanum phureja could be clearly differentiated from tetraploid (Solanum tuberosum) genotypes. Many of the proteins apparently contributing to genotype differentiation are involved in disease and defense responses, the glycolytic pathway, and sugar metabolism or protein targeting/storage. Only nine proteins out of 730 showed significant differences between GM lines and their controls. There was much less variation between GM lines and their non-GM controls compared with that found between different varieties and landraces. A number of proteins were identified by mass spectrometry and added to a potato tuber two-dimensional protein map.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Proteome analyses using accurate mass and elution time peptide tags with capillary LC time-of-flight mass spectrometry.

We describe the application of capillary liquid chromatography (LC) time-of-flight (TOF) mass spectrometric instrumentation for the rapid characterization of microbial proteomes. Previously (Lipton et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11049) the peptides from a series of growth conditions of Deinococcus radiodurans have been characterized using capillary LC MS/MS and accurate mass measurements which are captured as an accurate mass and time (AMT) tag database. Using this AMT tag database, detected peptides can be assigned using measurements obtained on a TOF due to the additional use of elution time data as a constraint. When peptide matches are obtained using AMT tags (i.e., using both constraints) unique matches of a mass spectral peak occurs 88% of the time. Not only are AMT tag matches unique in most cases, the coverage of the proteome is high; approximately 3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, approximately 900 ORFs detected using LC-TOFMS, with approximately 500 ORFs covered by at least two AMT tags. These results indicate that AMT database searches with modest mass and elution time criteria can provide proteomic information for approximately one thousand proteins in a single run of <3 h. The advantage of this method over using MS/MS based techniques is the large number of identifications that occur in a single experiment as well as the basis for improved quantitation. For MS/MS experiments, the number of peptide identifications is severely restricted because of the time required to dissociate the peptides individually. These results demonstrate the utility of the AMT tag approach using capillary LC-TOF MS instruments, and also show that AMT tags developed using other instrumentation can be effectively utilized.

Amino Acid Sequence↗
PubMed2006

Comparative proteome analysis of laboratory grown Brucella abortus 2308 and Brucella melitensis 16M.

Brucella species are pathogenic agents that cause brucellosis, a debilitating zoonotic disease that affects a large variety of domesticated animals and humans. Brucella melitensis and Brucella abortus are considered major health threats because of their highly infectious nature and worldwide occurrence. The availability of the annotated genomes for these two species has allowed a comparative proteomics study of laboratory grown B. melitensis 16M and B. abortus 2308 by two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting. Computer-assisted analysis of the different 2-D gel images of strains 16M and 2308 revealed significant quantitative and qualitative differences in their protein expression patterns. Proteins involved in membrane transport, particularly the high affinity amino acids binding proteins, and those involved in Sec-dependent secretion systems related to type IV and type V secretion systems, were differentially expressed. Differential expression of these proteins may be responsible for conferring specific host preference in the two strains 2308 and 16M.

Animals↗
PubMed2006

The proteins of human chromosome 21.

Recent genomic sequence annotation suggests that the long arm of human chromosome 21 encodes more than 400 genes. Because there is no evidence to exclude any significant segment of 21 q from containing genes relevant to the Down syndrome (DS) cognitive phenotype, all genes in this entire set must be considered as candidates. Only a subset, however, is likely to make critical contributions. Determining which these are is both a major focus in biology and a critical step in efficient development of therapeutics. The subtle molecular abnormality in DS, the 50% increase in chromosome 21 gene expression, presents significant challenges for researchers in detection and quantitation. Another challenge is the current limitation in understanding gene functions and in interpreting biological characteristics. Here, we review information on chromosome 21-encoded proteins compiled from the literature and from genomics and proteomics databases. For each protein, we summarize their evolutionary conservation, the complexity of their known protein interactions and their level of expression in brain, and discuss the implications and limitations of these data. For a subset, we discuss neurologically relevant phenotypes of mouse models that include knockouts, mutations, or overexpression. Lastly, we highlight a small number of genes for which recent evidence suggests a function in biochemical/cellular pathways that are relevant to cognition. Until knowledge deficits are overcome, we suggest that effective development of gene-phenotype correlations in DS requires a serious and continuous effort to assimilate broad categories of information on chromosome 21 genes, plus the creation of more versatile mouse models.

Animals↗
PubMed2026

Multiomics profiling of plasma reveals lipid-immune dysregulation and exosome remodeling in mpox and mpox-HIV co-infection.

BACKGROUND: Monkeypox virus (MPXV) infects diverse human cell types, and human immunodeficiency virus (HIV) co-infection is common. The immunometabolic consequences of MPXV infection, and how it may be altered by HIV, remain poorly defined. METHODS: We performed quantitative plasma lipidomics and precise metabolomics in a discovery cohort (n = 81) comprising MPXV-monoinfected (MPLWOH), MPXV-HIV-coinfected (MPLWH), and HIV-monoinfected (PLWH) patients and healthy controls, integrating exosome proteomics, cytokine profiling, and transcriptomics of exosome-treated HepG2 and A549 cells for functional interpretation. An independent validation cohort (n = 65) was used to assess cross-cohort reproducibility. FINDINGS: MPXV infection induced broad lipid remodeling, with elevations in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and reductions in phosphatidylcholine (PC), lysophospholipids, cholesteryl ester (CE), and exosomal lecithin-cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL). These lipid alterations were correlated with tissue injury markers and inflammatory cytokines. The MPLWH group exhibited more severe metabolic disruption, including marked sulfatide (SL) depletion, lower cholesterol and high-density lipoprotein cholesterol (HDL-c), and extensive rewiring of lipid-cytokine associations. SL depletion in MPLWH correlated with abundances of COPI-mediated retrograde trafficking proteins in exosomes. Transcriptomic profiling of exosome-treated cells provided functional validation: MPLWOH exosomes induced lipid metabolism and repair-associated epithelial programs, while MPLWH exosomes drove phospholipid remodeling and acute inflammatory and mucosal barrier-stress responses. CONCLUSIONS: MPXV infection reprograms host lipid metabolism and exosome composition, with HIV co-infection amplifying inflammatory, metabolic, and trafficking disruptions. These convergent multi-omics signatures link systemic lipid dysregulation to exosome-mediated immunomodulation and identify potential targets for host-directed interventions. FUNDING: This study was funded by the Major Project of Guangzhou National Laboratory.

Adult↗
PubMedDate not supplied

Murine retrovirus infection and the effect of chronic alcohol consumption: proteomic analysis of cardiac protein expression.

AIMS: The cardiovascular complications of acquired immunodeficiency syndrome (AIDS) are serious, including the occurrence of pathological heart conditions such as cardiomyopathy. Chronic alcohol consumption accentuates the severity of AIDS and may contribute to the development of cardiomyopathy. The aim of this work was to use a proteomics approach to investigate global alterations in protein expression in a mouse model of AIDS in the presence or absence of chronic alcohol consumption. METHODS: Cardiac proteins were separated by two-dimensional polyacrylamide gel electrophoresis and quantitative computer analysis was used to evaluate the resulting two-dimensional protein profiles. Proteins that were differentially expressed in the hearts of mice from the different experimental groups were identified by peptide mass finger-printing by matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: A number of specific proteins were observed to be differentially expressed in the mouse heart due to the effect of ethanol feeding alone. Differentially expressed proteins were also observed that were due to viral infection alone. Ethanol feeding and viral infection appeared to have similar effects on the expression of a number of proteins. A total of 24 proteins were altered by infection alone. Of these 24 proteins, eight were affected by alcohol, with six alterations being ameliorated and two being exacerbated by alcohol. Two of these proteins have been identified as the 27 kDa heat-shock protein and mitochondrial long-chain acyl-CoA thioesterase 1. CONCLUSIONS: These results suggest that chronic alcohol consumption may exacerbate the effects of viral infection on the heart by lowering the stress response leading to de-protection and further cytotoxic effects.

Administration, Oral↗
PubMedDate not supplied

Phage display antibody-based proteomic device using resonance-enhanced detection.

The combination of phage display antibody arrays with a novel nanotransducer technique based on resonant nanoparticles in a nanosandwiched film enables the sensitive parallel screening of proteins. Using the resonance of nanoparticles with their induced mirror dipoles in a thin-film structure, limitations of fluorophores, such as unspecific background and nonvisibility to the eye, can be overcome, thereby leading to an optical signal significantly more sensitive than that of standard colloid techniques. The signal can be both directly observed as a color change of a microdot at the sensor surface and tuned throughout the visible range of the spectrum. Here we report the application of an optical chip using scFv-antibody-antigen interactions. Artificial scFv-antibodies against a variety of proteins, including yeast enzymes and bovine serum albumin (as a standard), were constructed via Phage Display. These scFv-antibodies were then coated onto metal nanoclusters and bound to their antigens that were arrayed as nanodroplets at the resonance layer of the chip. ScFv-Antibody-antigen interaction resulted in a visible array of microdots. Using resonance-enhanced absorption, the absorption signal of the spots was amplified by one to two orders of magnitude (compared to colloid-based techniques). For quantitative analysis, either an 8-micron scanner or a CCD camera (resolution 4 microns) was employed to gain direct-reflection spectra rather than unspecific scatter data (prone to dust and unspecific interaction). Our results demonstrate that this device enables high-throughput proteomics to overcome some limitations of fluorescence, enzyme labels, and colloid techniques.

Antibodies↗
PubMed2005

Protein sequence tags: a novel solution for comparative proteomics.

Comparative proteome profiling using stable isotope peptide labelling and mass spectrometry has emerged as a promising strategy. Here, we show the broad potential of our proprietary protein sequence tag (PST) technology. A special feature of PST is its ability to detect a wide variety of proteins including the pharmaceutically relevant membrane and nuclear proteins. This procedure addresses a similar number of proteins, compared to the multidimensional protein identification technology approach, but offers additionally a quantitative analysis with its recently developed quantitative PST version.

Amino Acid Sequence↗
PubMed2002

Biomic study of human myeloid leukemia cells differentiation to macrophages using DNA array, proteomic, and bioinformatic analytical methods.

A biomic approach by integrating three independent methods, DNA microarray, proteomics and bioinformatics, is used to study the differentiation of human myeloid leukemia cell line HL-60 into macrophages when induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Analysis of gene expression changes at the RNA level using cDNA against an array of 6033 human genes showed that 5950 (98.6%) of the genes were expressed in the HL-60 cells. A total of 624 genes (10.5%) were found to be regulated during HL-60 cell differentiation. Most of these genes have not been previously associated with HL-60 cells and include genes encoded for secreted proteins as well as genes involved in cell adhesion, signaling transduction, and metabolism. Protein analysis using two-dimensional gel electrophoresis showed a total of 682 distinct protein spots; 136 spots (19.9%) exhibited quantitative changes between HL-60 control and macrophages. These differentially expressed proteins were identified by mass spectrometry. We developed a bioinformatics program, the Bulk Gene Search System (BGSS, http://www.sinica.edu.tw:8900/perl/genequery.pl) to search for the functions of genes and proteins identified by cDNA microarrays and proteomics. The identified regulated proteins and genes were classified into seven groups according to subcellular locations and functions. This powerful holistic biomic approach using cDNA microarray, proteomics coupled to bioinformatics can provide in-depth information on the impact and importance of the regulated genes and proteins for HL-60 differentiation.

Cell Differentiation↗
PubMed2006

Proteome resolution by two-dimensional gel electrophoresis varies with the commercial source of IPG strips.

By facilitating reproducible first dimension separations, commercial immobilized pH gradient (IPG) strips enable high throughput and high-resolution proteomic analyses using two-dimensional gel electrophoresis (2DE). Amersham, Biorad, Invitrogen, and Sigma all market linear pH 3-10 IPG strips. We have applied optimized 2DE protocols with both membrane and soluble brain protein extracts to critically evaluate all four products. Resolved protein spots were quantitatively evaluated after carrying out these protocols using IPG strips from the four companies. Biorad and Amersham IPG strips resolved a high number of membrane and soluble proteins, respectively. Furthermore, Amersham IPG strips eluted the largest amount of protein into the second dimension gels and had the most protein remaining in the strip after 2DE. Biorad and Amersham IPG strips maintained a consistent linear pH 3-10 gradient, whereas those from Invitrogen appeared nonlinear or "compressed" within the central pH region. The gradient range within Sigma IPG strips appeared to be slightly less than pH 3-10, due to one extended pH unit within the gradient. Overall, all four commercially available IPG strips have the ability to resolve both membrane and soluble brain proteomes. The difference is that Amersham and Biorad do so more consistently and with better spot resolution. It appears that the physical/chemical nature of commercially available IPG strips can vary considerably, leading to marked differences in subsequent protein resolution in 2DE. These differences likely reflect variations in the uptake of proteins into the strips, and differences in the focusing and elution of proteins from the first to the second dimension. These differences would appear, in part, to underlie some inter-lab variations in the effective resolution of proteomes.

Animals↗
PubMed2007

Proteomics discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP-2 substrate degradome.

Elucidation of protease substrate degradomes is essential for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell function. We identified matrix metalloproteinase-2 (MMP-2) cleaved proteins, solubilized pericellular matrix, and shed cellular ectodomains in the cellular context using a new multiplex proteomics approach. Tryptic peptides of intact and cleaved proteins, collected from conditioned culture medium of Mmp2(-/-) fibroblasts expressing low levels of transfected active human MMP-2 at different time points, were amine-labeled with iTRAQ mass tags. Peptide identification and relative quantitation between active and inactive protease transfectants were achieved following tag fragmentation during tandem MS. Known substrates of MMP-2 were identified thereby validating this technique with many novel MMP-2 substrates including the CX(3)CL1 chemokine fractalkine, osteopontin, galectin-1, and HSP90alpha also being identified and biochemically confirmed. In comparison with ICAT-labeling and quantitation, 8-9-fold more proteins and substrates were identified by iTRAQ. "Peptide mapping," the location of multiple peptides identified within a particular protein by iTRAQ in combination with their relative abundance ratios, enabled the domain shed and general location of the cleavage site to be identified in the native cellular substrate. Hence this advance in degradomics cell-based screens for native protein substrates casts new light on the roles for proteases in cell function.

Amino Acid Sequence↗
PubMed2004

Biological spectra analysis: Linking biological activity profiles to molecular structure.

Establishing quantitative relationships between molecular structure and broad biological effects has been a longstanding challenge in science. Currently, no method exists for forecasting broad biological activity profiles of medicinal agents even within narrow boundaries of structurally similar molecules. Starting from the premise that biological activity results from the capacity of small organic molecules to modulate the activity of the proteome, we set out to investigate whether descriptor sets could be developed for measuring and quantifying this molecular property. Using a 1,567-compound database, we show that percent inhibition values, determined at single high drug concentration in a battery of in vitro assays representing a cross section of the proteome, provide precise molecular property descriptors that identify the structure of molecules. When broad biological activity of molecules is represented in spectra form, organic molecules can be sorted by quantifying differences between biological spectra. Unlike traditional structure-activity relationship methods, sorting of molecules by using biospectra comparisons does not require knowledge of a molecule's putative drug targets. To illustrate this finding, we selected as starting point the biological activity spectra of clotrimazole and tioconazole because their putative target, lanosterol demethylase (CYP51), was not included in the bioassay array. Spectra similarity obtained through profile similarity measurements and hierarchical clustering provided an unbiased means for establishing quantitative relationships between chemical structures and biological activity spectra. This methodology, which we have termed biological spectra analysis, provides the capability not only of sorting molecules on the basis of biospectra similarity but also of predicting simultaneous interactions of new molecules with multiple proteins.

Molecular Structure↗
PubMed2006

Evaluation of 2-DE protein patterns from pre- and postnatal stages of the mouse brain.

Brains of the mouse from three developmental stages, embryo day 16 (Ed16), postnatal stage one week (1W) and eight weeks (8W), were distributed to different laboratories for a collaborative proteome analysis (The Human Brain Proteome Project). As one of the laboratories involved in this project, we separated total protein extracts of the brains by large gel 2-DE. From the 2-DE protein patterns a section was evaluated for each of the three stages according to resolution, reproducibility and quantitative changes using an image analysis software. The evaluated pattern section was selected to allow comparisons of 2-DE patterns between different laboratories on the basis of optimum separation. Changes in protein expression were analysed within two phases of development: Stage Ed16 versus stage 1W and stage 1W versus stage 8W. Out of the 200 protein spots evaluated 5-6% showed quantitative changes in the range of > or = 30% between two stages. The relationship in the frequency of up- and down-regulated protein spots differed between the two investigated phases. Most of the protein spots which showed altered expression between two stages were identified by MS. High quality in protein separation and evaluation is demonstrated.

Aging↗
PubMed2006

Protein clusters associated with carcinogenesis, histological differentiation and nodal metastasis in esophageal cancer.

We examined the proteomic background of esophageal cancer. We used laser microdissection to obtain tumor tissues from 72 esophageal squamous cell carcinoma cases and adjacent normal tissues in 57 of these cases. The 2D-DIGE generated quantitative expression profiles with 1730 protein spots. Based on the intensity of the protein spots, unsupervised classification distinguished the tumor tissues from their normal counterparts, and subdivided the tumor tissues according to their histological differentiation. We identified 498 protein spots with altered intensity in the tumor tissues, which protein identification by LC-MS/MS showed to correspond to 217 gene products. We also found 41 protein spots that were associated with nodal metastasis, and identified 33 proteins corresponding to the spots, including cancer-associated proteins such as alpha-actinin 4, hnRNP K, periplakin, squamous cell carcinoma antigen 1 and NudC. The identified cancer-associated proteins have been previously reported to be individually involved in a range of cancer types, and our study observed them collectively in a single type of malignancy, esophageal cancer. As the identified proteins are involved in important biological processes such as cytoskeletal/structural organization, transportation, chaperon, oxidoreduction, transcription and signal transduction, they may function in a coordinate manner in carcinogenesis and tumor progression of esophageal cancer.

Carcinoma, Squamous Cell↗