Faculty Bibliography

Isolating high-priority segments of genomes greatly enhances the efficiency of next-generation sequencing (NGS) by allowing researchers to focus on their regions of interest. For the 2010-11 DNA Sequencing Research Group (DSRG) study, we compared outcomes from two leading companies, Agilent Technologies (Santa Clara, CA, USA) and Roche NimbleGen (Madison, WI, USA), which offer custom-targeted genomic enrichment methods. Both companies were provided with the same genomic sample and challenged to capture identical genomic locations for DNA NGS. The target region totaled 3.5 Mb and included 31 individual genes and a 2-Mb contiguous interval. Each company was asked to design its best assay, perform the capture in replicates, and return the captured material to the DSRG-participating laboratories. Sequencing was performed in two different laboratories on Genome Analyzer IIx systems (Illumina, San Diego, CA, USA). Sequencing data were analyzed for sensitivity, specificity, and coverage of the desired regions. The success of the enrichment was highly dependent on the design of the capture probes. Overall, coverage variability was higher for the Agilent samples. As variant discovery is the ultimate goal for a typical targeted sequencing project, we compared samples for their ability to sequence single-nucleotide polymorphisms (SNPs) as a test of the ability to capture both chromosomes from the sample. In the targeted regions, we detected 2546 SNPs with the NimbleGen samples and 2071 with Agilent's. When limited to the regions that both companies included as baits, the number of SNPs was approximately 1000 for each, with Agilent and NimbleGen finding a small number of unique SNPs not found by the other.

Selected Reaction Monitoring (SRM) is a method of choice for accurate quantitation of low-abundance proteins in complex backgrounds. This strategy is, however, sensitive to interference from other components in the sample that have the same precursor and fragment masses as the monitored transitions. We present here an approach to detect interference by using the expected relative intensity of SRM transitions. We also designed an algorithm to automatically detect the linear range of calibration curves. These approaches were applied to the experimental data of Clinical Proteomic Tumor Analysis Consortium (CPTAC) Verification Work Group Study 7 and show that the corrected measurements provide more accurate quantitation than the uncorrected data.

ATP-sensitive K(+) (K(ATP) ) channels are expressed ubiquitously, but have diverse roles in various organs and cells. Their diversity can partly be explained by distinct tissue-specific compositions of four copies of the pore-forming inward rectifier potassium channel subunits (Kir6.1 and/or Kir6.2) and four regulatory sulfonylurea receptor subunits (SUR1 and/or SUR2). Channel function and/or subcellular localization also can be modified by the proteins with which they transiently or permanently interact to generate even more diversity. We performed a quantitative proteomic analysis of K(ATP) channel complexes in the heart, endothelium, insulin-secreting min6 cells (pancreatic beta-cell like), and the hypothalamus to identify proteins with which they interact in different tissues. Glycolysis is an overrepresented pathway in identified proteins of the heart, min6 cells, and the endothelium. Proteins with other energy metabolic functions were identified in the hypothalamic samples. These data suggest that the metabolo-electrical coupling conferred by K(ATP) channels is conferred partly by proteins with which they interact. A large number of identified cytoskeletal and trafficking proteins suggests endocytic recycling may help control K(ATP) channel surface density and/or subcellular localization. Overall, our data demonstrate that K(ATP) channels in different tissues may assemble with proteins having common functions, but that tissue-specific complex organization also occurs.

Nature uses a wide variety of protein post-translational modifications to regulate protein structure and activity and tandem mass spectrometry has emerged as the most powerful analytical approach to detect these moieties. However, modified peptides present special challenges for characterization. First, they are generally present at sub-stoichiometric levels, meaning that without enrichment strategies samples are dominated by unmodified peptides, so finding the modified peptides may be a challenge. Secondly, the modifications may have unique fragmentation behaviors in collision- induced dissociation (CID), which may need to be considered by database search engines. Finally, if there are multiple residues within a given peptide that could bear a particular modification type, then it is necessary to identify fragment ions that frame either side of the modification site in order to be able to localize the exact site of modification within the peptide. In this study the Proteome Informatics Research Group (iPRG) of the Association of Biomolecular Resource Facilities (ABRF) assessed participants' ability to find a variety of post-translationally modified peptides within a complex peptide mixture background. The dataset consisted of nearly twenty thousand high resolution and high mass accuracy tandem mass spectra. Within the sample there were peptides with a variety of different natural and chemical modifications. The data were provided in several formats and participants were required to submit their results in a provided Excel spreadsheet template, then complete a short survey to document their approach to data analysis. All submissions were anonymous. Overall spectrum identification performance was assessed, but particular emphasis was placed on their ability to detect and localize modifications of potential biological significance. A comparison of the different approaches and results achieved by all participants will be presented

Nature uses a wide variety of protein post-translational modifications to regulate protein structure and activity and tandem mass spectrometry has emerged as the most powerful analytical approach to detect these moieties. However, modified peptides present special challenges for characterization. First, they are generally present at sub-stoichiometric levels, meaning that without enrichment strategies samples are dominated by unmodified peptides, so finding the modified peptides may be a challenge. Secondly, the modifications may have unique fragmentation behaviors in collision-induced dissociation (CID), which may need to be considered by database search engines. Finally, if there are multiple residues within a given peptide that could bear a particular modification type, then it is necessary to identify fragment ions that frame either side of the modification site in order to be able to localize the exact site of modification within the peptide. In this study the Proteome Informatics Research Group (iPRG) of the Association of Biomolecular Resource Facilities (ABRF) assessed participants' ability to find a variety of posttranslationally modified peptides within a complex peptide mixture background. The dataset consisted of nearly twenty thousand high resolution and high mass accuracy tandem mass spectra. Within the sample there were peptides with a variety of different natural and chemical modifications. The data were provided in several formats and participants were required to submit their results in a provided Excel spreadsheet template, then complete a short survey to document their approach to data analysis. All submissions were anonymous. Overall spectrum identification performance was assessed, but particular emphasis was placed on their ability to detect and localize modifications of potential biological significance. A comparison of the different approaches and results achieved by all participants will be presented

The field of label-free biophysical technologies used to quantitatively characterize macromolecular interactions with each other and with small molecules has grown enormously in the last 10 years. The most widely used analytical technologies for characterizing biomolecular interactions are surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), biolayer interferometry (BLI), and analytical ultracentrifugation (AUC). Measuring interaction parameters accurately and quantitatively is challenging, as it requires specialized expertise, training, and instrumentation. The Molecular Interaction Research Group (MIRG) conducted an online survey designed to capture the current profile of label-free technologies, including ITC, SPR, and other biosensors used in academia and the pharmaceutical industry sector. The main goal of the survey was to take a snapshot of laboratory, instrumentation, applications for measuring various biophysical parameters, confidence in data interpretation, data validation and acceptability, and limitations of using various technologies. Through this survey, we anticipate that the participating laboratories will be able to gauge their own capabilities and gain insights into the relative success of the different technologies that they use for characterizing molecular interactions.

Cardiolipin is a dimeric phospholipid with a characteristic acyl composition that is generated by fatty acid remodeling after de novo synthesis. Several enzymes have been proposed to participate in acyl remodeling of cardiolipin. In order to compare the effect of these enzymes, we determined the pattern of cardiolipin molecular species in Drosophila strains with specific enzyme deletions, using MALDI-TOF mass spectrometry with internal standards. We established the linear range of the method for cardiolipin quantification, determined the relative signal intensities of several cardiolipin standards, and demonstrated satisfying signal-to-noise ratios in cardiolipin spectra from a single fly. Our data demonstrate changes in the cardiolipin composition during the Drosophila life cycle. Comparison of cardiolipin spectra, using vector algebra, showed that inactivation of tafazzin had a large effect on the molecular composition of cardiolipin, inactivation of calcium-independent phospholipase A(2) had a small effect, whereas inactivation of acyl-CoA:lysocardiolipin-acyltransferase and of the trifunctional enzyme did not affect the cardiolipin composition.

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4mu8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

The apical surface of mammalian bladder urothelium is covered by large (500-1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin-Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, resulting in the accumulation of the uroplakin-degrading multivesicular bodies. Finally, although MAL and uroplakins cofloat in detergent-resistant raft fractions, they are associated with distinct plaque and hinge membrane subdomains, respectively. These data suggest a model in which 1) MAL does not play a role in the apical sorting of uroplakins; 2) the propensity of uroplakins to polymerize forming 16-nm particles and later large 2D crystals that behave as detergent-resistant (giant) rafts may drive their apical targeting; 3) the exclusion of MAL from the expanding 2D crystals of uroplakins explains the selective association of MAL with the hinge areas in the uroplakin-delivering fusiform vesicles, as well as at the apical surface; and 4) the hinge-associated MAL may play a role in facilitating the incorporation of the exocytic uroplakin vesicles into the corresponding hinge areas of the urothelial apical surface.