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PubMed2006

Semiautomated high-sensitivity profiling of human blood serum glycoproteins through lectin preconcentration and multidimensional chromatography/tandem mass spectrometry.

We describe an effective analytical approach to identify trace glycoproteins in a small volume of human serum. The system is based on automatable affinity enrichment through silica-based lectin microcolumns and a further separation of the retained glycoproteins on a reversed-phase liquid chromatography with superficially porous packing, operating at high temperature. The fractionated sample is further directed into a 96-well plate for trypsinization and LC-MS/MS analysis. Using a major-component depleted blood serum (16 microg total protein), we were able to identify 271 glycoproteins through this analytical system.

Chromatography, Affinity↗
PubMed2006

Challenges related to analysis of protein spot volumes from two-dimensional gel electrophoresis as revealed by replicate gels.

Assumptions that need to be considered prior to statistical analysis of protein spot volumes from two-dimensional gel electrophoresis (2-DE) data are studied using replicate gels of the same sample. The most important observation is that the data tables of protein spot volumes from 2-DE images contain a large number of missing values, which are not consistent with the presence or absence of the proteins. This implies both loss of information and problems for the subsequent statistical analysis. Challenges with 2-DE protein spot volumes are viewed in light of multiple gel comparisons and multivariate data analysis.

Data Interpretation, Statistical↗
PubMed2005

Immunochemical and proteomic analysis of covalent adducts formed by quinone methide tumor promoters in mouse lung epithelial cell lines.

Two quinone methide (QM) metabolites of the phenolic antioxidant butylated hydroxytoluene (BHT), 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) and the tert-butyl-hydroxylated derivative (BHTOH-QM), are believed to be responsible for promoting lung tumor formation in mice treated with BHT. QMs are strongly electrophilic and undergo Michael type additions with nucleophiles at the exocyclic methylene to form benzylic thioether adducts. Our goal was to identify intracellular protein targets of these QMs in order to gain insight into their effects on tumorigenesis. Cell line E10 of mouse lung epithelial origin and its spontaneous transformant, the tumorigenic E9 cell line, were treated with BHT-QM or BHTOH-QM, and cellular proteins were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Adducted proteins were detected on western blots with polyclonal antibodies developed to a conjugate of BHTOH-QM that recognized adducts of both QMs bound to thiol groups of Cys and side chain amino groups of Lys and His residues. Tryptic digests of immunoreactive proteins were analyzed by HPLC mass spectrometry (LC/MS) and identified by searching protein databases using MS/MS data. In a few cases, adducted peptides in these digests were detected by matrix-assisted laser desorption/ionization time-of-flight MS. A total of 37 immunoreactive proteins were identified including proteins involved in carbohydrate metabolism, nucleic acid synthesis, and RNA and protein processing, in addition to several cytoskeletal and stress-related proteins. About half of the protein adducts were found in both cell lines. Adducts detected only in transformed E9 cells include glutathione S-transferase P1, peroxiredoxin 2, nucleoside diphosphate kinase, and vinculin, whereas several alkylated cytoskeletal proteins such as tubulins, vimentin, calvasculin, and calcyclin were detected exclusively in E10 cells. Several of the proteins modified by BHT-derived QMs have been implicated in various aspects of tumorigenesis and are excellent candidates for further study into the consequences of alkylation on cellular transformation.

Animals↗
PubMed2003

Construction of linear functional expression elements with DNA and RNA hybrid primers: a flexible and fast method for proteomics.

Two methods of constructing linear functional expression elements (LFEE) using hybrid DNA and RNA primers in DNA amplification for rapid gene expression are described. In both methods, it is not necessary to have additional transformation or bacterial propagation. The promoter, open reading frame (ORF) and terminator are amplified using Pfu or Taq DNA polymerase. Three elements containing DNA or RNA overhang are covalently ligated by T4 DNA ligase. The recombinant molecule is amplified with element-specific primers. The LFEE can be generated by both methods in a few hours and can be expressed in mammalian cells.

Animals↗
PubMed2004

Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli.

The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis. Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain. The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant. Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated. Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of aspartate, a key metabolic precursor of threonine. Synthesis of aspartase (aspartate ammonia-lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of aspartate to fumarate and ammonia. Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant. The far lower level of cystathionine beta-lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine. In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E. coli mutant.

Aspartic Acid↗
PubMed2004

A dataset of human liver proteins identified by protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry.

Proteins from human liver carcinoma Huh7 cells, representing transformed liver cells, and cultured primary human fetal hepatocytes (HFH) and human HH4 hepatocytes, representing nontransformed liver cells, were extracted and processed for proteome analysis. Proteins from stimulated cells (interferon-alpha treatment for the Huh7 and HFH cells and induction of hepatitis C virus [HCV] proteins for the HH4 cells) and corresponding control cells were labeled with light and heavy cleavable ICAT reagents, respectively. The labeled samples were combined, trypsinized, and subject to cation-exchange and avidin-affinity chromatographies. The resulting cysteine-containing peptides were analyzed by microcapillary LC-MS/MS. The MS/MS spectra were initially analyzed by searching the human International Protein Index database using the SEQUEST software (1). Subsequently, new statistical algorithms were applied to the collective SEQUEST search results of each experiment. First, the PeptideProphet software (2) was applied to discriminate true assignments of MS/MS spectra to peptide sequences from false assignments, to assign a probability value for each identified peptide, and to compute the sensitivity and error rate for the assignment of spectra to sequences in each experiment. Second, the ProteinProphet software (3) was used to infer the protein identifications and to compute probabilities that a protein had been correctly identified, based on the available peptide sequence evidence. The resulting protein lists were filtered by a ProteinProphet probability score p > or = 0.5, which corresponded to an error rate of less than 5%. A total of 1,296, 1,430, and 1,476 proteins or related protein groups were identified in three subdatasets from the Huh7, HFH, and HH4 cells, respectively. In total, these subdatasets contained 2,486 unique protein identifications from human liver cells. An increase of the threshold to p > or = 0.9 (corresponding to an error rate of less than 1%) resulted in 2,159 unique protein identifications (1,146, 1,235, and 1,318 for the Huh7, HFH, and HH4 cells, respectively).

Algorithms↗
PubMed2004

The human plasma proteome: a nonredundant list developed by combination of four separate sources.

We have merged four different views of the human plasma proteome, based on different methodologies, into a single nonredundant list of 1175 distinct gene products. The methodologies used were 1) literature search for proteins reported to occur in plasma or serum; 2) multidimensional chromatography of proteins followed by two-dimensional electrophoresis and mass spectroscopy (MS) identification of resolved proteins; 3) tryptic digestion and multidimensional chromatography of peptides followed by MS identification; and 4) tryptic digestion and multidimensional chromatography of peptides from low-molecular-mass plasma components followed by MS identification. Of 1,175 nonredundant gene products, 195 were included in more than one of the four input datasets. Only 46 appeared in all four. Predictions of signal sequence and transmembrane domain occurrence, as well as Genome Ontology annotation assignments, allowed characterization of the nonredundant list and comparison of the data sources. The "nonproteomic" literature (468 input proteins) is strongly biased toward signal sequence-containing extracellular proteins, while the three proteomics methods showed a much higher representation of cellular proteins, including nuclear, cytoplasmic, and kinesin complex proteins. Cytokines and protein hormones were almost completely absent from the proteomics data (presumably due to low abundance), while categories like DNA-binding proteins were almost entirely absent from the literature data (perhaps unexpected and therefore not sought). Most major categories of proteins in the human proteome are represented in plasma, with the distribution at successively deeper layers shifting from mostly extracellular to a distribution more like the whole (primarily cellular) proteome. The resulting nonredundant list confirms the presence of a number of interesting candidate marker proteins in plasma and serum.

Biomarkers, Tumor↗
PubMed2005

Protein function, connectivity, and duplicability in yeast.

Protein-protein interaction networks have evolved mainly through connectivity rewiring and gene duplication. However, how protein function influences these processes and how a network grows in time have not been well studied. Using protein-protein interaction data and genomic data from the budding yeast, we first examined whether there is a correlation between the age and connectivity of yeast proteins. A steady increase in connectivity with protein age is observed for yeast proteins except for those that can be traced back to Eubacteria. Second, we investigated whether protein connectivity and duplicability vary with gene function. We found a higher average duplicability for proteins interacting with external environments than for proteins localized within intracellular compartments. For example, proteins that function in the cell periphery (mainly transporters) show a high duplicability but are lowly connected. Conversely, proteins that function within the nucleus (e.g., transcription, RNA and DNA metabolisms, and ribosome biogenesis and assembly) are highly connected but have a low duplicability. Finally, we found a negative correlation between protein connectivity and duplicability.

Evolution, Molecular↗
PubMed2004

Recent developments in biomarkers for the early detection of lung cancer: perspectives based on publications 2003 to present.

PURPOSE OF REVIEW: Given the poor prognosis associated with lung cancer, the ability to diagnose lung cancer in its early stages is considered crucial to achieving decreased lung cancer mortality. Herein, we discuss recent advances in biomarker discovery and evaluation of their potential application in the clinical setting. RECENT FINDINGS: Novel potential biomarkers have been identified via new techniques including cDNA microarray analysis, comparative genomic hybridization, and proteomics. These factors have been evaluated in various validation studies including tissue microarrays, RT-PCR, and assays of bioactivity. The characterization of these potential biomarkers through analysis of pathways that have been associated with neo-plastic transformation may help to identify the precursor lesions that are associated with subsequent progression to invasive carcinoma. In addition, the past year has also produced intriguing results regarding the detection of biomarkers in easily accessible screening specimens such as sputa, serum, and exhaled breath. Recent advances in these aspects of biomarker identification for the early detection of lung cancer are reviewed. SUMMARY: Identification of new candidate biomarkers and improved applications of previously detected biomarkers show great promise for the ultimate establishment of practical lung cancer screening. While recent studies engender optimism for the creation of clinically applicable screening tests, the biomarkers that have been identified need larger, follow-up validational studies and further characterization as to their biologic importance.

Biomarkers, Tumor↗
PubMed2006

Plasticity-related regulation of the hippocampal proteome.

Plasticity of glutamatergic synapses is considered to be a pivotal mechanism underlying the ability of the CNS to re-configure its neural circuits. A large number of studies have focused on investigating how individual proteins, biochemical pathways and structural processes alter both the induction and maintenance of synaptic plasticity. However, it is likely that synaptic plasticity involves temporally and spatially coordinated regulation of multiple protein complexes within the activated neural circuit. By using a global proteomics-based approach we have now been able to reveal that highly diverse protein classes exhibit altered expression in response to both the activation of glutamate receptors and the induction of long-term potentiation (LTP) of glutamatergic synaptic strength in the hippocampus; a brain area where plastic synaptic modification is believed to be key to cognitive processes, such as spatial learning. Of the 2946 resolvable protein spots detected in this study, 79 (2.7%) were significantly altered in abundance in response to 100 microM glutamate application (all P < 0.05). The majority (56 out of 79) of these changes were due to the activation of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptor. Likewise, the induction of LTP was associated with an altered abundance of 2.4% of the detectable proteome during the early (10 min) phase and 1.7% during the late (4 h) phase of its development. Observed changes in temporal and protein class-specific patterns of expression depict a widespread shift from metabolic to structural protein alteration as the plasticity process matures.

2-Amino-5-phosphonovalerate↗
PubMed2006

Reticulons RTN3 and RTN4-B/C interact with BACE1 and inhibit its ability to produce amyloid beta-protein.

Beta-secretase beta-site APP cleaving enzyme 1 (BACE1), is a membrane-bound aspartyl protease necessary for the generation of amyloid beta-protein (Abeta), which accumulates in the brains of individuals with Alzheimer's disease (AD). To gain insight into the mechanisms by which BACE1 activity is regulated, we used proteomic methods to search for BACE1-interacting proteins in human neuroblastoma SH-SY5Y cells, which overexpress BACE1. We identified reticulon 4-B (RTN4-B; Nogo-B) as a BACE1-associated membrane protein. Co-immunoprecipitation experiments confirmed a physical association between BACE1 and RTN4-B, RTN4-C (the shortest isoform of RTN-4), and their homologue reticulon 3 (RTN3), both in SH-SY5Y cells and in transfected human embryonic kidney (HEK) 293 cells. Overexpression of these reticulons (RTNs) resulted in a 30-50% reduction in the secretion of both Abeta40 and Abeta42 from HEK293 cells expressing the AD-associated Swedish mutant amyloid precursor protein (APP), but did not affect Abeta secretion from cells expressing the APP beta-C-terminal fragment (beta-CTF), indicating that these RTNs can inhibit BACE1 activity. Furthermore, a BACE1 mutant lacking most of the N-terminal ectodomain also interacted with these RTNs, suggesting that the transmembrane region of BACE1 is critical for the interaction. We also observed a similar interaction between these RTNs and the BACE1 homologue BACE2. Because RTN3 and RTN4-B/C are substantially expressed in neural tissues, our findings suggest that they play important roles in the regulation of BACE1 function and Abeta production in the brain.

Amyloid Precursor Protein Secretases↗
PubMed2006

Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome.

Characterization of the composition of the postsynaptic proteome (PSP) provides a framework for understanding the overall organization and function of the synapse in normal and pathological conditions. We have identified 698 proteins from the postsynaptic terminal of mouse CNS synapses using a series of purification strategies and analysis by liquid chromatography tandem mass spectrometry and large-scale immunoblotting. Some 620 proteins were found in purified postsynaptic densities (PSDs), nine in AMPA-receptor immuno-purifications, 100 in isolates using an antibody against the NMDA receptor subunit NR1, and 170 by peptide-affinity purification of complexes with the C-terminus of NR2B. Together, the NR1 and NR2B complexes contain 186 proteins, collectively referred to as membrane-associated guanylate kinase-associated signalling complexes. We extracted data from six other synapse proteome experiments and combined these with our data to provide a consensus on the composition of the PSP. In total, 1124 proteins are present in the PSP, of which 466 were validated by their detection in two or more studies, forming what we have designated the Consensus PSD. These synapse proteome data sets offer a basis for future research in synaptic biology and will provide useful information in brain disease and mental disorder studies.

Animals↗
PubMed2006

Patatins, Kunitz protease inhibitors and other major proteins in tuber of potato cv. Kuras.

The major potato tuber proteins of the Kuras cultivar, which is the dominant cultivar used in Northern Europe for industrial starch production, were analysed using 1D and 2D gel electrophoresis. The electrophoretic patterns varied significantly depending on the method of preparation and the potato variant (Solanum tuberosum). Proteins were characterized using MS and scored against potato protein databases, derived from both 'Kuras only' and 'all potato' expressed sequence tags (EST) and full-length cDNAs. Despite the existence of approximately 180 000 ESTs, the currently available potato sequence data showed a severe under-representation of genes or long transcripts encoding proteins > 50 kDa (3.5% of all) compared with the complete proteome of Arabidopsis thaliana (33% of all). We found that patatin and Kunitz protease inhibitor (KPI) variants are extraordinarily dominant in Kuras tuber and, most significantly, that their amino acid sequences are specific to Kuras. Other proteins identified include annexin, glyoxalase I, enolase and two lipoxygenases, the sequences of which are highly conserved among potato variants. Known S. tuberosum patatins cluster into three clades all represented in Kuras. S. tuberosum KPIs cluster into more diverse clades of which five were found in Kuras tuber, including a novel clade, KPI K, found to date only in Kuras. Furthermore, protein abundance was contrasted with the levels of corresponding gene transcripts found in our previous EST and LongSAGE studies of Kuras tuber.

Amino Acid Sequence↗
PubMed2004

Amyloid beta-peptide(1-42) contributes to the oxidative stress and neurodegeneration found in Alzheimer disease brain.

Oxidative stress is extensive in Alzheimer disease (AD) brain. Amyloid beta-peptide (1-42) has been shown to induce oxidative stress and neurotoxicity in vitro and in vivo. Genetic mutations that result in increased production of Abeta1-42 from amyloid precursor protein are associated with an early onset and accelerated pathology of AD. Consequently, Abeta1-42 has been proposed to play a central role in the pathogenesis of AD as a mediator of oxidative stress. In this review, we discuss the role of Abeta1-42 in the lipid peroxidation and protein oxidation evident in AD brain and the implications of such oxidative stress for the function of various proteins that we have identified as specifically oxidized in AD brain compared to control, using proteomics methods. Additionally, we discuss the critical role of methionine 35 in the oxidative stress and neurotoxic properties exhibited by Abeta1-42.

Alzheimer Disease↗
PubMed2026

Small nucleolar RNA HIDDEN TREASURE 2 reduces drought tolerance via multiple pathways in Arabidopsis.

Small nucleolar RNAs (snoRNAs) contribute to ribosome biogenesis and modulate various aspects of plant growth and development. Given that osmotic stress downregulates numerous genes associated with ribosome biogenesis in roots, we hypothesize that snoRNAs might function in modulating plant responses to osmotic and drought stresses. To prove this hypothesis, we assessed the role of a C/D-box snoRNA, namely the HIDDEN TREASURE 2 (HID2), in Arabidopsis thaliana responses to drought using both loss-of-function and overexpression approaches. Under drought conditions, the Arabidopsis hid2 mutant displayed a significantly higher survival rate than both wild-type (WT) and HID2-complemented plants, while HID2-overexpressing plants showed a lower survival rate than WT. A series of physiological assays indicated that the hid2 mutant maintained a slower rate of water loss and more intact cell membranes than WT plants under drought, which supported their drought-tolerant phenotype. Comparative leaf transcriptome and proteome analyses revealed that processes related to wax biosynthesis, senescence, and anthocyanin accumulation were differentially regulated between hid2 and WT plants under water-deficit conditions. Consistently, the hid2 mutant accumulated higher amounts of wax and anthocyanins and exhibited delayed leaf senescence relative to WT plants under drought. Additionally, the hid2 mutant showed improved ability to increase sensitivity to abscisic acid (ABA), scavenge reactive oxygen species (ROS), and extended root hairs. Overall, these findings demonstrate HID2's role as a negative modulator in Arabidopsis drought tolerance by negatively affecting cell membrane stability, wax and anthocyanin biosynthesis, senescence, ROS-scavenging capacity, ABA responsiveness, and root hair formation.

Arabidopsis↗
PubMed2002

Plasticity of the kinomes in monkey and rat tissues.

The Kinetworks KPKS 1.0 immunoblotting screen was used to investigate the differential expression of 58 protein serine-threonine kinases, five protein threonine-tyrosine kinases, and 15 other nonreceptor protein tyrosine kinases in three types of human blood cells, 14 monkey organs, and six rat organs. The results demonstrate striking differences in the kineome profiles of diverse organs and even between the same organs in different species.

Animals↗
PubMed2004

Dissecting the human spliceosome through bioinformatics and proteomics approaches.

The precise excision of introns from mRNAs is executed by the spliceosome, a cellular machinery composed by five small nuclear RNAs and hundreds of proteins. In the last few years, several groups have used proteomics and computational biology tools to characterize the components of the human spliceosome. These reports have identified basically all known splicing factors and several new proteins. The composition of the human spliceosome confirms the link between splicing and other steps in gene expression. Here we comment on these reports and discuss the perspectives for the coming years.

Alternative Splicing↗
PubMed2005

Heat shock protein 27 is associated with freedom from graft vasculopathy after human cardiac transplantation.

Experimental studies have suggested that protective genes protect allografts from cardiac allograft vasculopathy (CAV), the major complication after cardiac transplantation. Here we have sought to confirm this hypothesis using long-term heart transplant recipients. Twenty-two patients that were 9 years or older after transplant were investigated; 11 of these were without angiographic evidence of CAV; 11 had developed early CAV at 1 to 3 years after transplant. To identify proteins that may act as protectors from CAV, a global proteomic approach was used comparing cardiac biopsies from 12 patients taken within the first 2 weeks after transplant and those taken after 9 years from the same patient. Proteins were separated by 2-D gel-electrophoresis, detected by silver staining, and analyzed using Progenesis software. A particular protein spot was found in 4/6 biopsies from patients without CAV, but absent from 5/6 biopsies from those with CAV (P=0.24); however, quantitative analysis of spot intensity showed a significant difference (0.061+/-0.05 versus 0.003+/-0.01, P=0.04). This spot was identified by mass spectrometry and a combination of techniques as a diphosphorylated form of HSP27. Immunohistochemistry of further biopsies not only validated that HSP27 was more abundantly expressed on biopsies without CAV but also showed it to be localized to blood vessels. In contrast, vessels from patients with CAV did not express HSP27 (P=0.028x10(-4)). Immunohistochemistry of 12 further early biopsies and nontransplanted heart showed HSP27 to be present in normal blood vessels. These findings suggest that expression of a specific diphosphorylated form of HSP27 is associated with healthy blood vessels; it appears to be lost from vessels of patients with graft vasculopathy.

Apoptosis↗