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David C Schriemer

Publications and source records attributed to David C Schriemer.

12 recordsLinked to original sources
PubMed2026

Nuclear body assembly by a viral repeat RNA promotes Kaposi's sarcoma-associated herpesvirus gene expression.

Kaposin is the most abundantly expressed viral RNA in tumors caused by the oncogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV); however, its role in viral replication is not understood. Here, we show that kaposin, previously viewed as a protein-coding transcript, exists primarily as a nuclear viral long non-coding RNA (lncRNA) that rebuilds cellular nuclear speckles (NSs) adjacent to the viral genome to enhance viral gene expression. Kaposin is both necessary and sufficient to drive substantial NS remodeling, and this effect depends on repetitive elements within the RNA. Absence of kaposin-mediated NS remodeling, depletion of the essential NS protein, serine/arginine repetitive matrix 2 (SRRM2), or steric blocking of the kaposin repetitive elements impair viral gene expression. This work defines kaposin as a viral architectural RNA that drives nuclear speckle seeding beside the viral genome and reframes our understanding of lncRNA function and the spatial organization of transcription in the infected cell nucleus.

Kaposi's sarcoma-associated herpesvirus↗
PubMed2006

Identification of trichloroethanol visualized proteins from two-dimensional polyacrylamide gels by mass spectrometry.

Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MS/MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC-MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of TCE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, TCE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. TCE visualized proteins are also shown to be well suited for protein identification using LC-MS/MS. In 16 spots selected for MS/MS analysis, TCE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, TCE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels.

Amino Acid Sequence↗
PubMed2006

Quantitating the statistical distribution of deuterium incorporation to extend the utility of H/D exchange MS data.

Measuring the statistical distribution of deuterium incorporated into enzymatically derived peptide fragments provides a valuable dimension to hydrogen/deuterium exchange mass spectrometry data. In this paper, we will discuss our improvement to the linear least-squares method for determining this distribution, through the addition of "zeroes" to the end of the deuterated isotopic envelope, to partially compensate for data truncation due to finite instrumental signal-to-noise ratios. The value of the distribution is demonstrated in a simple experimental example, where the linearity between average deuteration and percent D2O used to label test peptides hides a more complex relationship between the site-labeling probability and the total number of sites. This method offers the opportunity to resolve cases where a single peptide experiences distinct, independent biochemical states with each bearing a unique average deuteration; this can occur when a protein is modified to substoichiometric levels. From the experimentally determined distribution of a heterogeneously deuterated peptide, it was possible to extract the average deuteration of each component of the mixture.

Deuterium↗
PubMed2006

Detection and identification of sub-nanogram levels of protein in a nanoLC-trypsin-MS system.

Proteomic workflows involving liquid-based protein separations are an alternative to gel-based protein analysis, however the trypsin digestion procedure is usually difficult to implement, particularly when processing low abundance proteins from capillary column effluent. To convert the protein to peptides for the purpose of identification, current protocols require several sample handling steps, and sample losses become an issue. In this study, we present an improved system that conducts reversed-phase protein chromatography and rapid on-line tryptic digestion requiring sub-nanogram quantities of protein. This system employs a novel mirror-gradient concept that allows for dynamic titration of the column effluent to create optimal conditions for real-time tryptic digestion. The purpose behind this development was to improve the limits of detection of the online concept, to support flow-based alternatives to gel-based proteomics and to simplify the characterization of low abundance proteins. Using test mixtures of proteins, we show that peptide mass fingerprinting with high sequence representation can be easily achieved at the 20 fmol level, with detection limits down to 5 fmol (85 pg myoglobin). Limits of identification using standard data-dependent MS/MS experiments are as low as 10 fmol. These results suggest that the nanoLC-trypsin-MS/MS system could represent an alternative to the conventional "1D-gel to MS" proteomic strategy.

Amino Acid Sequence↗
PubMed2005

High-throughput screening for enzyme inhibitors using frontal affinity chromatography with liquid chromatography and mass spectrometry.

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated Kd value of 0.3 microM. Other binders yielded Kd values ranging from 0.35 to 3.35 microM. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.

Chromatography, Affinity↗
PubMed2005

Blending protein separation and peptide analysis through real-time proteolytic digestion.

Typical liquid- or gel-based protein separations require enzymatic digestion as an important first step in generating protein identifications. Traditional protocols involve long-term proteolytic digestion of the separated protein, often leading to sample loss and reduced sensitivity. Previously, we presented a rapid method of proteolytic digestion that showed excellent digestion of resistant and low concentrations of protein without requiring reduction and alkylation. Here, we demonstrate on-line, real-time tryptic digestion in conjunction with reversed-phase protein separation. The studies were aimed at optimizing pH and ionic strength and the size of the digestion element, to produce maximal protein digestion with minimal effects on chromatographic integrity. Upon establishing optimal conditions, the digestion element was attached downstream from a capillary C4 reversed-phase column. A four-protein mixture was processed through the combined system, and the resulting peptides were analyzed on-line by electrospray mass spectrometry. Extracted ion chromatograms for protein chromatography based on peptide elution were generated. These were shown to emulate ion chromatograms produced in a subsequent run without the digestion element, based on protein elution. The methodology will enable rapid and sensitive analysis of liquid-based protein separations using the power of bottom-up proteomics methodologies.

Chromatography, Liquid↗
PubMed2005

Emerging challenges in ligand discovery: new opportunities for chromatographic assay.

Ligand discovery initiatives are facing interesting challenges as ever-increasing numbers of proteins are entering screening programs. As an answer to steady pressure to improve performance in drug discovery, ligand discovery can expect to play an expanded role in generating small molecules as probes to help uncover the function of novel proteins. Chromatographic assay formats can offer new entry points into standard interaction characterization (binding and rate constants) as well as powerful, scaleable methods for compound screening. This review presents recent advancements in chromatographic assay technology, with a particular focus on frontal affinity chromatography as a platform technology for interaction analysis.

Chromatography↗
PubMed2003

Hydrogen/deuterium exchange mass spectrometry of actin in various biochemical contexts.

Hydrogen/deuterium exchange mass spectrometry (H/D MS) of monomeric actin (G-actin), polymeric actin (F-actin), phalloidin-bound F-actin and G-actin complexed with DNase I provides new insights into the architecture of F-actin and the effects of phalloidin and DNase I binding. Although the overall pattern of deuteration change supports the gross features of the Holmes F-actin model, two important differences were observed. Most significantly, no change in deuteration was observed in the critical "hydrophobic plug" region, suggesting this feature may not be present. Polymerization also produced deuteration increases for peptide fragments containing the ATP phosphate-binding loops, suggesting G-actin transitions to a more "open" conformation upon polymerization. However, polymerization produced decreases in deuteration mainly localized to the "inner", filament-axis side as predicted by the Holmes model. Mapping the phalloidin-induced decreases in F-actin deuteration onto the Lorenz binding site produced a single common patch straddling two monomers across the 1-start helix contact, again consistent with the Holmes architecture. Finally, both DNase I and phalloidin were able to alter the deuteration of regions distal to their respective binding sites. These results highlight the great opportunities for H/D MS to exploit high-resolution structures for detailed studies of the organization and dynamics of complex molecular assemblies.

Actins↗
PubMed2003

Frontal affinity chromatography-mass spectrometry assay technology for multiple stages of drug discovery: applications of a chromatographic biosensor.

This article presents new concepts in affinity chromatography/mass spectrometry for the study of molecular interactions. Chromatographic assays involving estrogen receptor-beta, sorbitol dehydrogenase, human alpha-thrombin, cholera toxin B subunit, beta-galactosidase, and Griffonia simplicifolia isolectin B(4) were established in microaffinity columns and operated in frontal analysis mode. Methods and formalism are presented for the measurement of dissociation constants, using direct methods in which the mass spectrometric signature of the ligand is used to measure breakthrough time and, hence, binding strength. The direct approach is capable of measuring sub-micromolar K(d) and higher, on sub-pmol amounts of immobilized protein, as shown in the cholera toxin assay. Indirect assays that demonstrate the advantage of routine, rugged performance were developed. By tracking the effect of a test ligand on a selected probe, or indicator ligand, dissociation constants in the low nanomolar range could be reliably determined for ligands to estrogen receptor-beta. Mass spectrometry supports the resolution of complex ligand mixtures, and it is demonstrated in the sorbitol dehydrogenase assay that ligands can be rank ordered across approximately three orders of magnitude in K(d), in a single run. A new concept for rapid mixture prescreening is presented, in which an indicator ligand can be used to discriminate between mixtures that contain high levels of weak ligands and those that contain single strong ligands.

Biosensing Techniques↗
PubMed2003

On-column digestion of proteins in aqueous-organic solvents.

Proteolytic digestion is an important step in protein identification by peptide mass mapping and tandem mass spectrometry (MS/MS)-based peptide sequencing. Traditional methods of protein digestion require extended incubation times and have difficulty with proteolytically resistant proteins. Here, we describe a method in which a protein solution was combined with a mixed aqueous-organic solution (methanol, isopropanol, or acetonitrile) and passed through a microcolumn containing immobilized trypsin. Myoglobin sequence coverage was high (>85%) in all three solvents, and differences in spectra were seen among the different solution conditions. Notably, methanol-based digestions produced fewer missed cleavages while acetonitrile-based digestions produced the most peptides and the most intense mass spectra. Flow rates through the column were varied from 0.5 to 15 micro L/min, corresponding to column residence times of 78 and 2.6 s, respectively. All flow rates produced high sequence coverage of myoglobin, although, at higher flow rates, more missed cleavages were observed. No significant increase in undigested myoglobin was observed with flow rates up to 15 micro L/min. The described method was applied to the digestion of human transferrin (hTf), a proteolytically resistant protein. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis detected 42 peptides covering 46% of the hTf sequence. The traditional aqueous method resulted in 12 peptides (8% sequence coverage) only when high concentrations of trypsin were used. Lastly, digestion of low nanomolar myoglobin was shown to produce detectable peptides and resulted in a correct database hit. Thus, we demonstrate a method that is capable of rapid on-line digestion, thereby lending itself to high-throughput identification of proteins.

Humans↗
PubMed2003

Function of oxygen resistance proteins in the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough.

Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H(2)O(2)) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H(2)O(2) resistance may also be conferred by two other rbr genes in the D. vulgaris genome. Inhibition of Sod activity by azide and H(2)O(2), but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D. vulgaris. Thus, Sor plays a key role in oxygen defense of D. vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.

Anaerobiosis↗