Project description:Phosphopeptide enrichment from complicated peptide mixtures is an essential step for mass spectrometry-based phosphoproteomic studies to reduce sample complexity and ionization suppression effects. Typical methods for enriching phosphopeptides include immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) beads, which have selective affinity and can interact with phosphopeptides. In this study, the IMAC enrichment method was compared with the TiO2 enrichment method, using a multistep enrichment strategy from whole cell lysate, to evaluate their abilities to enrich for different types of phosphopeptides. The peptide-to-beads ratios were optimized for both IMAC and TiO2 beads. Both IMAC and TiO2 enrichments were performed for three rounds to enable the maximum extraction of phosphopeptides from the whole cell lysates. The phosphopeptides that are unique to IMAC enrichment, unique to TiO2 enrichment, and identified with both IMAC and TiO2 enrichment were analyzed for their characteristics. Both IMAC and TiO2 enriched similar amounts of phosphopeptides with comparable enrichment efficiency. However, phosphopeptides that are unique to IMAC enrichment showed a higher percentage of multiphosphopeptides as well as a higher percentage of longer, basic, and hydrophilic phosphopeptides. Also, the IMAC and TiO2 procedures clearly enriched phosphopeptides with different motifs. Finally, further enriching with two rounds of TiO2 from the supernatant after IMAC enrichment or further enriching with two rounds of IMAC from the supernatant TiO2 enrichment does not fully recover the phosphopeptides that are not identified with the corresponding multistep enrichment.
Project description:Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening conditions. Furthermore a marked improvement in the correlation was found by using kinase constructs containing the catalytic domain in presence of additional domains or subunits.
Project description:Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3'-positions. As reported previously [Ahmadian,A., Gharizadeh,B., O'Meara,D., Odeberg,J. and Lundeberg,J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 5' end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.
Project description:Abstract: Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, methodologies to enrich the heterogeneous types of phosphopeptides are critical towards comprehensive mapping of the under-explored phosphoproteome. Taking advantage of the distinctive binding affinity of Ga3+ and Fe3+ towards phosphopeptides, we designed a tip-based metal-directed immobilized metal ion affinity (MD-IMAC) chromatography for sequential enrichment of phosphopeptides. On the analysis of Raji B cell, this sequential Ga3+-Fe3+-IMAC strategy demonstrated 1.5-3.5 fold superior phosphoproteomic coverage compared to the single IMAC (Fe3+, Ti4+, Ga3+ and Al3+). In addition, as high as 92% among the 6283 phosphopeptides was uniquely enriched by either 1st Ga3+-IMAC fraction (41%) or 2nd Fe3+-IMAC fraction (51%). The complementary property of Ga3+ and Fe3+ was further shown on the exclusively superior efficiency to enriched almost all the 1214 multiply phosphorylated peptides (99.4%) by 1st Ga3+-IMAC, while as low as 10% of 5069 monophosphorylated phosphopeptides was commonly enriched by both fractions. Application of our sequential Ga3+-Fe3+-IMAC approach to a human lung cancer tissue allowed the identification of 2560 unique phosphopeptides with only 8% overlapping. The fractionation ability was shown not only on the mono phosphopeptides and multiply phosphopeptides but also on the basic and acidic phosphopeptides; acidiphilic phosphorylation sites were predominately present in 1st Ga3+-IMAC (72%) and 85% Pro-directed and 79% basophilic phosphorylation sites were enriched by 2nd Fe3+-IMAC. Most interestingly, this strategy complementarily mapped different kinase substrate on the protein as well as site levels in multiple cellular pathways related to cancer invasion and metastasis of lung cancer ., Given the demonstrated fractionation ability, reproducibility, sensitivity and ease of tip preparation, we hope that this Ga3+-Fe3+-IMAC allow more comprehensive characterization of phosphoproteome in vitro and in vivo. Database search: The raw MS/MS data obtained by TripleTOF 5600 were processed using AB_SCIEX MS Data Converter with default parameters. All MS/MS files were analyzed using Mascot (Matrix Science, London, UK; version 2.3) against the SwissPort database (version 57.8) with the following constraints: an allowance for tryptic peptides of up to two missed cleavage sites, a fragment ion mass tolerance of 0.05 Da, and a parent ion tolerance of 10 ppm. Phosphorylation (S, T, Y) and oxidation (M) were selected as variable modifications. Searching on a randomized decoy database created by Mascot was required to evaluate the false discovery rate associated with protein identification. The false discovery rates with a Mascot score (p< 0.05) ranged between 0% and 1% in this study.
Project description:hESC have morphologic, genetic and genomic alternatiions when cells cultured in different passaging condition. Here transcriptome of four different hESC lines were compared in two passaging methods.
Project description:Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (K(D)) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte?Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand for the binding pocket of interest is known.
Project description:Understanding how to tune enzymatic activity is important not only for biotechnological applications, but also to elucidate the basic principles guiding the design and optimization of biological systems in nature. So far, the Michaelis-Menten equation has provided a fundamental framework of enzymatic activity. However, there is still no concrete guideline on how the parameters should be optimized towards higher activity. Here, we demonstrate that tuning the Michaelis-Menten constant ([Formula: see text]) to the substrate concentration ([Formula: see text]) enhances enzymatic activity. This guideline ([Formula: see text]) was obtained mathematically by assuming that thermodynamically favorable reactions have higher rate constants, and that the total driving force is fixed. Due to the generality of these thermodynamic considerations, we propose [Formula: see text] as a general concept to enhance enzymatic activity. Our bioinformatic analysis reveals that the [Formula: see text] and in vivo substrate concentrations are consistent across a dataset of approximately 1000 enzymes, suggesting that even natural selection follows the principle [Formula: see text].
Project description:It has been suggested that pathway analysis can complement single-SNP analysis in exploring genomewide association data. Pathway analysis incorporates the available biological knowledge of genes and SNPs and is expected to improve the chances of revealing the underlying genetic architecture of complex traits. Methods for pathway analysis can be classified as competitive (enrichment) or self-contained (association) according to the hypothesis tested. Although association tests are statistically more powerful than enrichment tests they can be difficult to calibrate because biases in analysis accumulate across multiple SNPs or genes. Furthermore, enrichment tests can be more scientifically relevant than association tests, as they detect pathways with relatively more evidence for association than the remaining genes. Here we show how some well known association tests can be simply adapted to test for enrichment, and compare their performance to some established enrichment tests. We propose versions of the Adaptive Rank Truncated Product (ARTP), Tail Strength Measure and Fisher's combination of p-values for testing the enrichment null hypothesis. We compare the behaviour of these proposed methods with the established Hypergeometric Test and Gene-Set Enrichment Analysis (GSEA). The results of the simulation study show that the modified version of the ARTP method has generally the best performance across the situations considered. The methods were also applied for finding enriched pathways for body mass index (BMI) and platelet function phenotypes. The pathway analysis of BMI identified the Vasoactive Intestinal Peptide pathway as significantly associated with BMI. This pathway has been previously reported as associated with BMI and the risk of obesity. The ARTP method was the method that identified the largest number of enriched pathways across all tested pathway databases and phenotypes. The simulation and data application results are in agreement with previous work on association tests and suggests that the ARTP should be preferred for both enrichment and association testing.
Project description:Multiple sequence alignments (MSAs) are a prerequisite for a wide variety of evolutionary analyses. Published assessments and benchmark data sets for protein and, to a lesser extent, global nucleotide MSAs are available, but less effort has been made to establish benchmarks in the more general problem of whole-genome alignment (WGA). Using the same model as the successful Assemblathon competitions, we organized a competitive evaluation in which teams submitted their alignments and then assessments were performed collectively after all the submissions were received. Three data sets were used: Two were simulated and based on primate and mammalian phylogenies, and one was comprised of 20 real fly genomes. In total, 35 submissions were assessed, submitted by 10 teams using 12 different alignment pipelines. We found agreement between independent simulation-based and statistical assessments, indicating that there are substantial accuracy differences between contemporary alignment tools. We saw considerable differences in the alignment quality of differently annotated regions and found that few tools aligned the duplications analyzed. We found that many tools worked well at shorter evolutionary distances, but fewer performed competitively at longer distances. We provide all data sets, submissions, and assessment programs for further study and provide, as a resource for future benchmarking, a convenient repository of code and data for reproducing the simulation assessments.