Search

In chromosome 11, 71 out of its 1254 proteins remain functionally uncharacterized on the basis of their existence evidence (uPE1s) following the latest version of neXtProt (release 2020-01-17). Because in vivo and in vitro experimental strategies are often time-consuming and labor-intensive, there is a need for a bioinformatics tool to predict the function annotation. Here, we used I-TASSER/COFACTOR provided on the neXtProt web site, which predicts gene ontology (GO) terms based on the 3D structure of the protein. I-TASSER/COFACTOR predicted 2413 GO terms with a benchmark dataset of the 22 proteins belonging to PE1 of chromosome 11. In this study, we developed a filtering algorithm in order to select specific GO terms using the GO map generated by I-TASSER/COFACTOR. As a result, 187 specific GO terms showed a higher average precision-recall score at the least cellular component term compared to 2413 predicted GO terms. Next, we applied 65 proteins belonging to uPE1s of chromosome 11, and then 409 out of 6684 GO terms survived, where 103 and 142 GO terms of molecular function and biological process, respectively, were included. Representatively, the cellular component GO terms of CCDC90B, C11orf52, and the SMAP were predicted and validated using the overexpression system into 293T cells and immunofluorescence staining. We will further study their biological and molecular functions toward the goal of the neXt-CP50 project as a part of C-HPP. We shared all results and programs in Github (https://github.com/heeyounh/I-TASSER-COFACTOR-filtering.git).

Human Proteome Project aims to map all human proteins including missing proteins as well as proteoforms with post translational modifications, alternative splicing variants (ASVs), and single amino acid variants (SAAVs). neXtProt and Ensemble databases are usually used to provide curated information on human coding genes. However, to find these proteoforms, we (Chr #11 team) first introduce a streamlined pipeline using customized and concatenated neXtProt and GENCODE originated from Ensemble, with controlled false discovery rate (FDR). Because of large sized databases used in this pipeline, we found more stringent FDR filtering (0.1% at the peptide level and 1% at the protein level) to claim novel findings, such as GENCODE ASVs and missing proteins, from human hippocampus data set (MSV000081385) and ProteomeXchange (PXD007166). Using our next generation proteomic pipeline (nextPP) with neXtProt and GENCODE databases, two missing proteins such as activity-regulated cytoskeleton-associated protein (ARC, Chr 8) and glutamate receptor ionotropic, kainite 5 (GRIK5, Chr 19) were additionally identified with two or more unique peptides from human brain tissues. Additionally, by applying the pipeline to human brain related data sets such as cortex (PXD000067 and PXD000561), spinal cord, and fetal brain (PXD000561), seven GENCODE ASVs such as ACTN4-012 (Chr.19), DPYSL2-005 (Chr.8), MPRIP-003 (Chr.17), NCAM1-013 (Chr.11), EPB41L1-017 (Chr.20), AGAP1-004 (Chr.2), and CPNE5-005 (Chr.6) were identified from two or more data sets. The identified peptides of GENCODE ASVs were mapped onto novel exon insertions, alternative translations at 5'-untranslated region, or novel protein coding sequence. Applying the pipeline to male reproductive organ related data sets, 52 GENCODE ASVs were identified from two testis (PXD000561 and PXD002179) and a spermatozoa (PXD003947) data sets. Four out of 52 GENCODE ASVs such as RAB11FIP5-008 (Chr. 2), RP13-347D8.7-001 (Chr. X), PRDX4-002 (Chr. X), and RP11-666A8.13-001 (Chr. 17) were identified in all of the three samples.

In the Chromosome-Centric Human Proteome Project (C-HPP), false-positive identification by peptide spectrum matches (PSMs) after database searches is a major issue for proteogenomic studies using liquid-chromatography and mass-spectrometry-based large proteomic profiling. Here we developed a simple strategy for protein identification, with a controlled false discovery rate (FDR) at the protein level, using an integrated proteomic pipeline (IPP) that consists of four engrailed steps as follows. First, using three different search engines, SEQUEST, MASCOT, and MS-GF+, individual proteomic searches were performed against the neXtProt database. Second, the search results from the PSMs were combined using statistical evaluation tools including DTASelect and Percolator. Third, the peptide search scores were converted into E-scores normalized using an in-house program. Last, ProteinInferencer was used to filter the proteins containing two or more peptides with a controlled FDR of 1.0% at the protein level. Finally, we compared the performance of the IPP to a conventional proteomic pipeline (CPP) for protein identification using a controlled FDR of <1% at the protein level. Using the IPP, a total of 5756 proteins (vs 4453 using the CPP) including 477 alternative splicing variants (vs 182 using the CPP) were identified from human hippocampal tissue. In addition, a total of 10 missing proteins (vs 7 using the CPP) were identified with two or more unique peptides, and their tryptic peptides were validated using MS/MS spectral pattern from a repository database or their corresponding synthetic peptides. This study shows that the IPP effectively improved the identification of proteins, including alternative splicing variants and missing proteins, in human hippocampal tissues for the C-HPP. All RAW files used in this study were deposited in ProteomeXchange (PXD000395).

Approximately 2.9 billion long base-pair human reference genome sequences are known to encode some 20 000 representative proteins. However, 3000 proteins, that is, ~15% of all proteins, have no or very weak proteomic evidence and are still missing. Missing proteins may be present in rare samples in very low abundance or be only temporarily expressed, causing problems in their detection and protein profiling. In particular, some technical limitations cause missing proteins to remain unassigned. For example, current mass spectrometry techniques have high limits and error rates for the detection of complex biological samples. An insufficient proteome coverage in a reference sequence database and spectral library also raises major issues. Thus, the development of a better strategy that results in greater sensitivity and accuracy in the search for missing proteins is necessary. To this end, we used a new strategy, which combines a reference spectral library search and a simulated spectral library search, to identify missing proteins. We built the human iRefSPL, which contains the original human reference spectral library and additional peptide sequence-spectrum match entries from other species. We also constructed the human simSPL, which contains the simulated spectra of 173 907 human tryptic peptides determined by MassAnalyzer (version 2.3.1). To prove the enhanced analytical performance of the combination of the human iRefSPL and simSPL methods for the identification of missing proteins, we attempted to reanalyze the placental tissue data set (PXD000754). The data from each experiment were analyzed using PeptideProphet, and the results were combined using iProphet. For the quality control, we applied the class-specific false-discovery rate filtering method. All of the results were filtered at a false-discovery rate of <1% at the peptide and protein levels. The quality-controlled results were then cross-checked with the neXtProt DB (2014-09-19 release). The two spectral libraries, iRefSPL and simSPL, were designed to ensure no overlap of the proteome coverage. They were shown to be complementary to spectral library searching and significantly increased the number of matches. From this trial, 12 new missing proteins were identified that passed the following criterion: at least 2 peptides of 7 or more amino acids in length or one of 9 or more amino acids in length with one or more unique sequences. Thus, the iRefSPL and simSPL combination can be used to help identify peptides that have not been detected by conventional sequence database searches with improved sensitivity and a low error rate.

The goal of the Chromosome-Centric Human Proteome Project (C-HPP) is to fully provide proteomic information from each human chromosome, including novel proteoforms, such as novel protein-coding variants expressed from noncoding genomic regions, alternative splicing variants (ASVs), and single amino acid variants (SAAVs). In the 144 LC/MS/MS raw files from human hippocampal tissues of control, epilepsy, and Alzheimer's disease, we identified the novel proteoforms with a workflow including integrated proteomic pipeline using three different search engines, MASCOT, SEQUEST, and MS-GF+. With a <1% false discovery rate (FDR) at the protein level, the 11 detected peptides mapped to four translated long noncoding RNA variants against the customized databases of GENCODE lncRNA, which also mapped to coding-proteins at different chromosomal sites. We also identified four novel ASVs against the customized databases of GENCODE transcript. The target peptides from the variants were validated by tandem MS fragmentation pattern from their corresponding synthetic peptides. Additionally, a total of 128 SAAVs paired with their wild-type peptides were identified with FDR <1% at the peptide level using a customized database from neXtProt including nonsynonymous single nucleotide polymorphism (nsSNP) information. Among these results, several novel variants related in neuro-degenerative disease were identified using the workflow that could be applicable to C-HPP studies. All raw files used in this study were deposited in ProteomeXchange (PXD000395).

A short and scalable synthesis of naamidine A, a marine alkaloid with a selective ability to inhibit epidermal growth factor receptor (EGFR)-dependent cellular proliferation, has been achieved. A key achievement in this synthesis was the development of a regioselective hydroamination of a monoprotected propargylguanidine to deliver N(3)-protected cyclic ene-guanidines. This permits the extension of this methodology to prepare N(2)-acyl analogues in a fashion that obviates the troublesome acylation of the free 2-aminoimidazoles, which typically yields mixtures of N(2)- and N(2),N(2)-diacylated products., Competing Interests: Notes The authors declare no competing financial interest.

A cascade silver(I)-catalyzed hydroamination/Michael addition sequence has been developed to deliver highly substituted bicyclic guanidines. This transformation gives rise to geometrically and constitutionally stable ene-guanidines and generates a remote stereocenter with moderate to high diastereoselectivity.

Human chromosome 11 is the third gene-rich chromosome having 1304 protein-coding genes. According to the GeneCards, this chromosome contains 240 genes related to diseases, as it is well known as a disease-rich chromosome. Although there are many protein-coding genes, the proteomic identification ratio is rather low. As a model study, human hippocampal tissues from patients suffering from Alzheimer's disease and epilepsy were prepared to evaluate the gene-centric statistics related to the gene expression and disorders of chromosome 11. A total of 8828 protein coding genes from brain tissues were extensively off-gel fractionated and profiled by a high resolution mass spectrometer with collision induced dissociation and electron transfer dissociation. Five-hundred twenty-three of the proteins from brain tissues were determined to belong to chromosome 11, representing 37% of the proteins reported in the Global Proteome Machine Database. We extracted gene clusters from a specific biological process or molecular function in gene ontology, among which the olfactory receptor genes showed the largest cluster on chromosome 11. Analysis of the proteome data set from the hippocampus provides a significant network associated with genes and proteins and leads to new insights into the biological and genetic mechanisms of chromosome 11-specific diseases such as Alzheimer's disease.

As the desired feature size of mold-assisted lithography decreases rapidly efficient demolding process becomes more challenging due to strong adhesion between polymeric resists and fine-featured molds. We synthesized new macromolecular additives and investigated the effects of surface energy and contraction of resist materials on demolding propensity by monitoring the adhesion force between the resist and the applied mold. The resist's surface energy was controlled, as inferred from water contact angle measurements, by chemically modifying its hydroxyl functionality. The resist's degree of volume shrinkage during the photocuring procedure was also controlled by mixing in a newly developed chemical that has a multiple radical chain transfer capability. The adhesion force was proportionally reduced as the surface energy of the resist materials decreased and as the volume shrinkage was reduced. When the volume shrinkage control was applied in conjunction with the low surface energy resist material (LS-30UV), we obtained an optimized condition requiring a minimum force for releasing the mold from the cured resist layer.

A well-defined cationic Ru-H complex catalyzes the dehydrative C-H alkylation reaction of phenols with alcohols to form ortho-substituted phenol products. Benzofuran derivatives are efficiently synthesized from the dehydrative C-H alkenylation and annulation reaction of phenols with 1,2-diols. The catalytic C-H coupling method employs cheaply available phenols and alcohols, exhibits a broad substrate scope, tolerates carbonyl and amine functional groups, and liberates water as the only byproduct., (© 2012 American Chemical Society)

The cationic ruthenium-hydride complex [(C(6)H(6))(PCy(3))(CO)RuH](+)BF(4) (-) (1) was found to be a highly effective catalyst for the intermolecular conjugate addition of simple alkenes to α,β-unsaturated carbonyl compounds to give (Z)-selective tetrasubstituted olefin products. The analogous coupling reaction of cinnamides with electron-deficient olefins led to the oxidative coupling of two olefinic C-H bonds in forming (E)-selective diene products. The intramolecular version of the coupling reaction efficiently produced indene and bicyclic fulvene derivatives. The empirical rate law for the coupling reaction of ethyl cinnamate with propene was determined as: rate = k[1](1)[propene](0)[cinnamate](-1). A negligible deuterium kinetic isotope effect (k(H)/k(D) = 1.1±0.1) was measured from both (E)-C(6)H(5)CH=C(CH(3))CONHCH(3) and (E)-C(6)H(5)CD=C(CH(3))CONHCH(3) with styrene. In contrast, a significant normal isotope effect (k(H)/k(D) = 1.7±0.1) was observed from the reaction of (E)-C(6)H(5)CH=C(CH(3))CONHCH(3) with styrene and styrene-d(10). A pronounced carbon isotope effect was measured from the coupling reaction of (E)-C(6)H(5)CH=CHCO(2)Et with propene ((13)C(recovered)/(13)C(virgin) at C(β) = 1.019(6)), while a negligible carbon isotope effect ((13)C(recovered)/(13)C(virgin) at C(β) = 0.999(4)) was obtained from the reaction of (E)-C(6)H(5)CH=C(CH(3))CONHCH(3) with styrene. Hammett plots from the correlation of para-substituted p-X-C(6)H(4)CH=CHCO(2)Et (X = OCH(3), CH(3), H, F, Cl, CO(2)Me, CF(3)) with propene and from the treatment of (E)-C(6)H(5)CH=CHCO(2)Et with a series of para-substituted styrenes p-Y-C(6)H(4)CH=CH(2) (Y = OCH(3), CH(3), H, F, Cl, CF(3)) gave the positive slopes for both cases (ρ = +1.1±0.1 and +1.5±0.1, respectively). Eyring analysis of the coupling reaction led to the thermodynamic parameters, Δ H(‡) = 20±2 kcal mol(-1) and S(‡) = -42±5 e.u. Two separate mechanistic pathways for the coupling reaction have been proposed on the basis of these kinetic and spectroscopic studies.

Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and iron-uptake transporters appeared to be dependent on tetracycline conditions, whereas β-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii.

The cationic ruthenium-hydride complex [(η(6)-C(6)H(6))(PCy(3))(CO)RuH](+)BF(4) (-) was found to be a highly regioselective catalyst for the oxidative C-H coupling reaction of aryl-substituted amides and unactivated alkenes to give ortho-alkenylamide products. The kinetic and spectroscopic analyses support a mechanism involving a rapid vinyl C-H activation followed by a rate-limiting C-C bond formation steps.

On the basis of the structural information that the host material has excellent charge stabilization, blue-emitting Ca(6-x-y)Mg(x)(PO(4))(4):Eu(y)(2+) (CMP:Eu(2+)) phosphors were synthesized and systematically optimized, and their photoluminescence (PL) properties were evaluated. Depending upon the amount of Mg added, the emission efficiency of the phosphors could be enhanced. The substitution of Eu(2+) affected their maximum wavelength (lambda(max)) and thermal stability because the substitution site of Eu(2+) could be varied. To obtain single-phase two-color-emitting phosphors, we incorporated Mn(2+) into CMP:Eu(2+) phosphors. Weak red emission resulting from the forbidden transition of Mn(2+) could be enhanced by the energy transfer from Eu(2+) to Mn(2+) that occurs because of the spectral overlap between the photoluminescence excitation (PLE) spectrum of Mn(2+) and the PL spectrum of Eu(2+). The energy transfer process was confirmed by the luminescence spectra, energy transfer efficiency, and decay curve of the phosphors. Finally, the optimized Ca(6-x-y)Mg(x-z)(PO(4))(4):Eu(y)(2+),Mn(z)(2+) (CMP:Eu(2+),Mn(2+)) phosphors were applied with green emitting Ca(2)MgSi(2)O(7):Eu(2+) (CMS:Eu(2+)) phosphors to ultraviolet (UV) light emitting diode (LED)-pumped white LEDs. The CMS:Eu(2+)-mixed CMP:Eu(2+), Mn(2+)-based white LEDs showed an excellent color rendering index (CRI) of 98 because of the broader emission band and more stable color coordinates than those of commercial Y(3)Al(5)O(12):Ce(3+) (YAG:Ce(3+))-based white LEDs under a forward bias current of 20 mA. The fabricated white LEDs showed very bright natural white light that had the color coordinate of (0.3288, 0.3401), and thus CMP:Eu(2+),Mn(2+) could be regarded as a good candidate for UV LED-based white LEDs.

Conjugated linoleic acid (CLA) production by rumen bacteria is closely related to biohydrogenation of linoleic acid (LA) and affected by various environmental factors. Ruminal biohydrogenation and isomerization were characterized in view of incubation conditions using a mixed culture of microorganisms obtained from surgically prepared cows. Free-floating bacteria (FFB) produced more CLA than particle-attached bacteria (PAB). Some major factors affecting the ruminal environment such as diet, concentrations of fat substrates, incubation time, pre-incubation, and the presence of glucose in the medium were found to be important determinants for the ruminal production of CLA and in a close relationship with biohydrogenation. The mixed bacterial culture, which was pre-exposed to LA, produced more CLA than an unexposed control in a medium containing 30% rumen fluid. The rate of conversion of fat substrate (LA) to hydrogenated products (trans-C18:1, C18:0) was negatively correlated with the initial LA concentration. Overall, the present study showed that CLA accumulation can be increased by modification of diet-induced environmental conditions, which affect changes in ruminal microflora.

The cationic complex [(pymox-Me(2))RuCl(2)](+)BF(4)(-) was found to be a highly effective catalyst for the C-H bond oxidation reaction of arylalkanes in water. For example, the treatment of ethylbenzene (1.0 mmol) with t-BuOOH (3.0 mmol) and 1.0 mol % of the Ru catalyst in water (3 mL) cleanly produced PhCOCH(3) at room temperature. Both a large kinetic isotope effect (k(H)/k(D) = 14) and a relatively large Hammett value (rho = -1.1) suggest a solvent-caged oxygen rebounding mechanism via a Ru(IV)-oxo intermediate species.

Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC-MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.