Jonathan A. Epstein

De Novo Peptide Sequencing Using Exhaustive Enumeration of Peptide Composition

We introduce the use of a peptide composition lookup table indexed by residual mass and number of amino acids for de novo sequencing of polypeptides. Polypeptides of 1600 Daltons (Da) or more can be sequenced effectively through exhaustive compositional analysis of MS/MS spectra obtained by unimolecular decomposition (without CID) in a MALDI TOF/TOF despite a fragment mass accuracy of 50 mDa. Peaks are referenced against the lookup table to obtain a complete profile of amino acid combinations, and combinations are assembled into series of increasing length. Concatenating the differences between successive entries in compositional series yields peptide sequences that can be scored and ranked according to signal intensity. While the current work involves measurements acquired on MALDI TOF-TOF, such general treatment of the data anticipates extension to other types of mass analyzers.

Comparative whole genome sequencing reveals phenotypic tRNA gene duplication in spontaneous Schizosaccharomyces pombe La mutants

We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNASerUCA-C47:6U with a debilitating substitution in its variable arm fails to produce tRNA in a sla1-rrm mutant deficient for RNA chaperone-like activity. The parent strain and spontaneous mutant were analyzed using Solexa sequencing. One synonymous single-nucleotide polymorphism (SNP), unrelated to the phenotype, was identified. Further sequence analyses found a duplication of the tRNASerUCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNASerUCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNASerUCA-C47:6U levels in sla1-rrm but not sla1-null cells, consistent with non-specific low-affinity interactions contributing to the RNA chaperone-like activity of La, similar to other RNA chaperones. Our analysis also identified 24 SNPs between ours and S. pombe 972h- strain yFS101 that was recently sequenced using Solexa. By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a ‘reference’ mtDNA, providing the first identification of these S. pombe mtDNA discrepancies. Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing.

Imprecise transcription termination within Escherichia coli greA leader gives rise to an array of short transcripts, GraL

We report that greA expression is driven by two strong, overlapping P1 and P2 promoters. The P1 promoter is σ70-dependent and P2 is σE-dependent. Two-thirds of transcripts terminate within the leader region and the remaining third comprises greA mRNA. Termination efficiency seems to be unaffected by growth phase. Two collections of small 40–50 (initiating from P2) and 50–60 nt (from P1) RNA chains, termed GraL, are demonstrable in vivo and in vitro. We document that GraL arrays arise from an intrinsic terminator with an 11 bp stem followed by an AU7GCU2 sequence. Atypical chain termination occurs at multiple sites; the 3′-ends differ by 1 nt over a range of 10 nt. Transcripts observed are shown to be insensitive to Gre factors and physically released from RNAP–DNA complexes. The abundance of individual chains within each cluster displays a characteristic pattern, which can be differentially altered by oligonucleotide probes. Multiple termination sites are particularly sensitive to changes at the bottom of the stem. Evolutionarily conserved GraL stem structures and fitness assays suggest a biological function for the RNA clusters themselves. Although GraL overexpression induces ≥3-fold transcriptional changes of over 100 genes, a direct target remains elusive.

SNPfisher: tools for probing genetic variation in laboratory-reared zebrafish

Single nucleotide polymorphisms (SNPs) are the benchmark molecular markers for modern genomics. Until recently, relatively few SNPs were known in the zebrafish genome. The use of next-generation sequencing for the positional cloning of zebrafish mutations has increased the number of known SNP positions dramatically. Still, the identified SNP variants remain under-utilized, owing to scant annotation of strain specificity and allele frequency. To address these limitations, we surveyed SNP variation in three common laboratory zebrafish strains using whole-genome sequencing. This survey identified an average of 5.04 million SNPs per strain compared with the Zv9 reference genome sequence. By comparing the three strains, 2.7 million variants were found to be strain specific, whereas the remaining variants were shared among all (2.3 million) or some of the strains. We also demonstrate the broad usefulness of our identified variants by validating most in independent populations of the same laboratory strains. We have made all of the identified SNPs accessible through ‘SNPfisher’, a searchable online database (snpfisher.nichd.nih.gov). The SNPfisher website includes the SNPfisher Variant Reporter tool, which provides the genomic position, alternate allele read frequency, strain specificity, restriction enzyme recognition site changes and flanking primers for all SNPs and Indels in a user-defined gene or region of the zebrafish genome. The SNPfisher site also contains links to display our SNP data in the UCSC genome browser. The SNPfisher tools will facilitate the use of SNP variation in zebrafish research as well as vertebrate genome evolution. SUMMARY: SNP variation in three common laboratory zebrafish strains is analyzed using whole-genome sequencing and is organized into a user-friendly and searchable online database.

Production of Reliable MALDI Spectra with Quality Threshold Clustering of Replicates

We present the first application of the quality threshold (QT) clustering algorithm to mass spectrometry (MS) data. The unique abilities of QT clustering to yield precision nodes that are commensurate with the mass measurement precision of the instrument are exploited to generate a consensus spectrum out of multiple replicate spectra. The spectral dot product and confidence intervals are used as a tool for evaluating the similarity and reproducibility between the consensus and replicates. The method is equally applicable to high and low resolution measurements. This paper demonstrates applications to linear spectra from a matrix assisted laser desorption ionization (MALDI) time of flight (TOF) instrument as well as peptide fragmentation data obtained from a TOF/TOF after unimolecular decomposition. The advantages of clustering to mitigate the inherent precision the shortcomings of MALDI data are discussed.

Phosphodiesterase sequence variants may predispose to prostate cancer

We hypothesized that mutations that inactivate phosphodiesterase (PDE) activity and lead to increased cAMP and cyclic guanosine monophosphate levels may be associated with prostate cancer (PCa). We sequenced the entire PDE coding sequences in the DNA of 16 biopsy samples from PCa patients. Novel mutations were confirmed in the somatic or germline state by Sanger sequencing. Data were then compared to the 1000 Genome Project. PDE, CREB and pCREB protein expression was also studied in all samples, in both normal and abnormal tissue, by immunofluorescence. We identified three previously described PDE sequence variants that were significantly more frequent in PCa. Four novel sequence variations, one each in the PDE4B,PDE6C, PDE7B and PDE10A genes, respectively, were also found in the PCa samples. Interestingly, PDE10A and PDE4B novel variants that were present in 19 and 6% of the patients were found in the tumor tissue only. In patients carrying PDE defects, there was pCREB accumulation (P<0.001), and an increase of the pCREB:CREB ratio (patients 0.97±0.03; controls 0.52±0.03; P-value <0.001) by immunohistochemical analysis. We conclude that PDE sequence variants may play a role in the predisposition and/or progression to PCa at the germline and/or somatic state respectively.

De novo sequencing of peptides

Publisher Summary De novo sequencing involves deducing a peptides sequence purely from mass spectral fragmentation data. There are roughly two ways to view the relationship between de novo sequencing and database search algorithms. First is complementary, and the second is alternative. Approaches that emphasize the complementary relationship between de novo sequences and database searches treat de novo sequences as a means to enhance the quality and reliability of database searches. Regimes originating from this approach utilize the spectra to derive one or more highly reliable sequence tags. These tags guide the database matching process, and since they ostensibly represent the most prominent and reliable features of the spectra, the tags also impart a higher degree of confidence to the database results. De novo sequencing approaches also differ according to the way in which they determine the fragment type (y or b) and score the sequence information. These differences in approach tend to generate great differences between de novo sequences obtained even from the same spectra. There are two principal approaches to the implementation of partial de novo sequencing called the global and local paradigms.

ProteinProcessor: A probabilistic analysis using mass accuracy and the MS spectrum.

Current approaches to protein identification rely heavily on database matching of fragmentation spectra or precursor peptide ions. We have developed a method for MALDI TOF‐TOF instrumentation that uses peptide masses and their measurement errors to confirm protein identifications from a first pass MS/MS database search. The method uses MS1‐level spectral data that have heretofore been ignored by most search engines. This approach uses the distribution of mass errors of peptide matches in the MS1 spectrum to develop a probability model that is independent of the MS/MS database search identifications. Peptide mass matches can come from both precursor ions that have been fragmented as well as those that are tentatively identified by accurate mass alone. This additional corroboration enables us to confirm protein identifications to MS/MS‐based scores that are otherwise considered to be only of moderate quality. Straightforward and easily applicable to current proteomic analyses, this tool termed “ProteinProcessor” provides a robust and invaluable addition to current protein identification tools.