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Nusinow, David

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Nusinow

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David

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Nusinow, David

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2014 - 20173

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Author's Publications: search.filters.isAuthorOfPublication.e3fb35fb-b74d-4f7f-87bf-e836f803878a

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    Estimating the Selective Effects of Heterozygous Protein Truncating Variants from Human Exome Data
    (2017) Cassa, Christopher; Weghorn, Donate; Balick, Daniel; Jordan, Daniel M.; Nusinow, David; Samocha, Kaitlin E.; O’Donnell-Luria, Anne; MacArthur, Daniel; Daly, Mark; Beier, David R.; Sunyaev, Shamil
    The dispensability of individual genes for viability has interested generations of geneticists. For some genes it is essential to maintain two functional chromosomal copies, while others may tolerate the loss of one or both copies. Exome sequence data from 60,706 individuals provide sufficient observations of rare protein truncating variants (PTVs) to make genome-wide estimates of selection against heterozygous loss of gene function. The cumulative frequency of rare deleterious PTVs is primarily determined by the balance between incoming mutations and purifying selection rather than genetic drift. This enables the estimation of the genome-wide distribution of selection coefficients for heterozygous PTVs and corresponding Bayesian estimates for individual genes. The strength of selection can discriminate the severity, age of onset, and mode of inheritance in Mendelian exome sequencing cases. We find that genes under the strongest selection are enriched in embryonic lethal mouse knockouts, putatively cell-essential genes, Mendelian disease genes, and regulators of transcription. Screening by essentiality, we find a large set of genes under strong selection that likely have critical function but have not yet been extensively annotated in published literature.
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    An ultra-tolerant database search reveals that a myriad of modified peptides contributes to unassigned spectra in shotgun proteomics
    (2015) Chick, Joel M.; Kolippakkam, Deepak; Nusinow, David; Zhai, Bo; Rad, Ramin; Huttlin, Edward; Gygi, Steven
    Fewer than half of all tandem mass spectrometry (MS/MS) spectra acquired in shotgun proteomics experiments are typically matched to a peptide with high confidence. Here we determine the identity of unassigned peptides using an ultra-tolerant Sequest database search that allows peptide matching even with modifications of unknown masses up to ±500 Da. In a proteome-wide dataset on HEK293 cells (9,513 proteins and 396,736 peptides), this approach matched an additional 184,000 modified peptides, which were linked to biological and chemical modifications representing 523 distinct mass bins, including phosphorylation, glycosylation, and methylation. We localized all unknown modification masses to specific regions within a peptide. Known modifications were assigned to the correct amino acids with frequencies often >90%. We conclude that at least one third of unassigned spectra arise from peptides with substoichiometric modifications.
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    MultiNotch MS3 Enables Accurate, Sensitive, and Multiplexed Detection of Differential Expression across Cancer Cell Line Proteomes
    (American Chemical Society, 2014) McAlister, Graeme C.; Nusinow, David; Jedrychowski, Mark; Wühr, Martin; Huttlin, Edward; Erickson, Brian; Rad, Ramin; Haas, Wilhelm; Gygi, Steven
    Multiplexed quantitation via isobaric chemical tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)) has the potential to revolutionize quantitative proteomics. However, until recently the utility of these tags was questionable due to reporter ion ratio distortion resulting from fragmentation of coisolated interfering species. These interfering signals can be negated through additional gas-phase manipulations (e.g., MS/MS/MS (MS3) and proton-transfer reactions (PTR)). These methods, however, have a significant sensitivity penalty. Using isolation waveforms with multiple frequency notches (i.e., synchronous precursor selection, SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby increasing the number of reporter ions in the MS3 spectrum 10-fold over the standard MS3 method (i.e., MultiNotch MS3). By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements. To demonstrate utility, we analyzed biological triplicates of eight colon cancer cell lines using the MultiNotch MS3 method. Across all the replicates we quantified 8 378 proteins in union and 6 168 proteins in common. Taking into account that each of these quantified proteins contains eight distinct cell-line measurements, this data set encompasses 174 704 quantitative ratios each measured in triplicate across the biological replicates. Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments.