LAHEDES: the LAGLIDADG homing endonuclease database and engineering server.

LAGLIDADG homing endonucleases (LHEs) are DNA cleaving enzymes, also termed 'meganucleases' that are employed as gene-targeting reagents. This use of LHEs requires that their DNA specificity be altered to match sequences in genomic targets. The choice of the most appropriate LHE to target a particular gene is facilitated by the growing number of such enzymes with well-characterized activities and structures. 'LAHEDES' (The LAGLIDADG Homing Endonuclease Database and Engineering Server) provides both an online archive of LHEs with validated DNA cleavage specificities and DNA-binding interactions, as well as a tool for the identification of DNA sequences that might be targeted by various LHEs. Searches can be performed using four separate scoring algorithms and user-defined choices of LHE scaffolds. The webserver subsequently provides information regarding clusters of amino acids that should be interrogated during engineering and selection experiments. The webserver is fully open access and can be found at http://homingendonuclease.net.

Structural, functional and evolutionary relationships between homing endonucleases and proteins from their host organisms.

Homing endonucleases (HEs) are highly specific DNA-cleaving enzymes that are encoded by invasive DNA elements (usually mobile introns or inteins) within the genomes of phage, bacteria, archea, protista and eukaryotic organelles. Six unique structural HE families, that collectively span four distinct nuclease catalytic motifs, have been characterized to date. Members of each family display structural homology and functional relationships to a wide variety of proteins from various organisms. The biological functions of those proteins are highly disparate and include non-specific DNA-degradation enzymes, restriction endonucleases, DNA-repair enzymes, resolvases, intron splicing factors and transcription factors. These relationships suggest that modern day HEs share common ancestors with proteins involved in genome fidelity, maintenance and gene expression. This review summarizes the results of structural studies of HEs and corresponding proteins from host organisms that have illustrated the manner in which these factors are related.

Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family.

Novel family of putative homing endonuclease genes was recently discovered during analyses of metagenomic and genomic sequence data. One such protein is encoded within a group I intron that resides in the recA gene of the Bacillus thuringiensis 03058-36 bacteriophage. Named I-Bth0305I, the endonuclease cleaves a DNA target in the uninterrupted recA gene at a position immediately adjacent to the intron insertion site. The enzyme displays a multidomain, homodimeric architecture and footprints a DNA region of ~60 bp. Its highest specificity corresponds to a 14-bp pseudopalindromic sequence that is directly centered across the DNA cleavage site. Unlike many homing endonucleases, the specificity profile of the enzyme is evenly distributed across much of its target site, such that few single base pair substitutions cause a significant decrease in cleavage activity. A crystal structure of its C-terminal domain confirms a nuclease fold that is homologous to very short patch repair (Vsr) endonucleases. The domain architecture and DNA recognition profile displayed by I-Bth0305I, which is the prototype of a homing lineage that we term the 'EDxHD' family, are distinct from previously characterized homing endonucleases.

New structural proteins of Halobacterium salinarum gas vesicle revealed by comparative proteomics analysis.

The Halobacterium salinarum gas vesicle (GV) is an extremely stable intracellular organelle with air trapped inside a proteinaceous membrane. Reported here is a comparative proteomics analysis of GV and GV depleted lysate (GVD) to reveal the membrane structural proteins. Ten proteins encoded by gvp-1 (gvpMLKJIHGFED-1 and gvpACNO-1) and five proteins encoded by gvp-2 (gvpMLKJIHGFED-2 and gvpACNO-2) gene clusters for the biogenesis of spindle- and cylindrical-, respectively, shaped GV were identified by LC-MS/MS. The peptides of GvpA1, I1, J1, A2, and J2 were exclusively identified in purified GV, GvpD1, H1, L1, and F2 only in GVD, and GvpC1, N1, O1, F1, H2, and O2 in both samples. The identification of GvpA1, C1, F1, J1, and A2 in GV is in agreement with their previously known structural function. In addition, the detection of GvpI1, N1, O1, H2, J2, and O2 in GV suggested they are new structural proteins. Among these, the structural role of GvpI1 and N1 in GV was further validated by immuno-detection of protein A-tagged GvpI1 and N1 fusion proteins in purified GV. Thus, LC-MS/MS could reveal at least a half dozen gas vesicle structural proteins in the predominant spindle-shaped GV that may be helpful for studying its biogenesis.

Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs.

Site-specific homing endonucleases are capable of inducing gene conversion via homologous recombination. Reprogramming their cleavage specificities allows the targeting of specific biological sites for gene correction or conversion. We used computational protein design to alter the cleavage specificity of I-MsoI for three contiguous base pair substitutions, resulting in an endonuclease whose activity and specificity for its new site rival that of wild-type I-MsoI for the original site. Concerted design for all simultaneous substitutions was more successful than a modular approach against individual substitutions, highlighting the importance of context-dependent redesign and optimization of protein-DNA interactions. We then used computational design based on the crystal structure of the designed complex, which revealed significant unanticipated shifts in DNA conformation, to create an endonuclease that specifically cleaves a site with four contiguous base pair substitutions. Our results demonstrate that specificity switches for multiple concerted base pair substitutions can be computationally designed, and that iteration between design and structure determination provides a route to large scale reprogramming of specificity.

On the benefits of acquiring peptide fragment ions at high measured mass accuracy.

The advantages and disadvantages of acquiring tandem mass spectra by collision-induced dissociation (CID) of peptides in linear ion trap Fourier-transform hybrid instruments are described. These instruments offer the possibility to transfer fragment ions from the linear ion trap to the FT-based analyzer for analysis with both high resolution and high mass accuracy. In addition, performing CID during the transfer of ions from the linear ion trap (LTQ) to the FT analyzer is also possible in instruments containing an additional collision cell (i.e., the "C-trap" in the LTQ-Orbitrap), resulting in tandem mass spectra over the full m/z range and not limited by the ejection q value of the LTQ. Our results show that these scan modes have lower duty cycles than tandem mass spectra acquired in the LTQ with nominal mass resolution, and typically result in fewer peptide identifications during data-dependent analysis of complex samples. However, the higher measured mass accuracy and resolution provides more specificity and hence provides a lower false positive ratio for the same number of true positives during database search of peptide tandem mass spectra. In addition, the search for modified and unexpected peptides is greatly facilitated with this data acquisition mode. It is therefore concluded that acquisition of tandem mass spectral data with high measured mass accuracy and resolution is a competitive alternative to "classical" data acquisition strategies, especially in situations of complex searches from large databases, searches for modified peptides, or for peptides resulting from unspecific cleavages.

Genome-specific gas-phase fractionation strategy for improved shotgun proteomic profiling of proteotypic peptides.

Gas-phase fractionation (GPF) is an efficient and straightforward method to increase proteome coverage. In this report, optimal m/z ranges were calculated based on genomic complexity and experimental data. Then, theoretical precursor ion densities were calculated in silico from various organisms' genomes and found to corroborate the empirical selection of m/z ranges based on ion density mapping. According to both calculations, the choice of m/z range for most efficient GPF coverage in the lower m/z range should be very narrow and increase as m/z value increases. Next, a systematic LC-MS/MS analysis was performed to confirm this observation. The behavior of data-dependent precursor ion selection and the origin of the observed variability was investigated under three different scan modes of an LTQ-Orbitrap hybrid mass spectrometer. Finally, GPF combined with data-dependent analysis was compared to a targeted, pseudo-multiple reaction monitoring analysis of proteotypic peptides that should be, based on empirical observation of LC-ESI-MS/MS data, detectable. The result of the latter experiment supported our conclusion that data-dependent analysis using rational gas-phase fractionation was sufficient for comprehensive proteomic analysis of the proteotypic peptides in an unfractionated cell lysate.

ProSight PTM: an integrated environment for protein identification and characterization by top-down mass spectrometry.

ProSight PTM (https://prosightptm.scs.uiuc.edu/) is a web application for identification and characterization of proteins using mass spectra data from 'top-down' fragmentation of intact protein ions (i.e. without any tryptic digestion). ProSight PTM has many tools and graphical features to facilitate analysis of single proteins, proteins in mixtures and proteins fragmented in parallel. Sequence databases from across the phylogenetic tree are supported, with a new database strategy of 'shotgun annotation' used to assist characterization of wild-type proteins. During a database search, data from divergent sources regarding potential mass differences such as polymorphisms, alternate splicing and post-translational modifications are utilized. The user can optionally control how much of this biological variability should be searched.

Shotgun annotation of histone modifications: a new approach for streamlined characterization of proteins by top down mass spectrometry.

Eukaryotic histones serve as prototypical examples of posttranslational complexity with diverse modifications (PTMs) on many different residues that comprise a "Histone Code". To help crack this code more efficiently, we demonstrate a new strategy for protein characterization wherein complete PTM descriptions are obtained by database retrieval instead of manual interpretation of information-rich data from high-resolution tandem mass spectrometry (MS/MS). A database of nearly 50 000 modified histone H4 sequences was created and queried with 91 fragment ions from electron capture dissociation of a histone form +112 Da (versus unmodified mass) selectively accumulated in a quadrupole Fourier transform hybrid mass spectrometer. The correct form atop the retrieval list indicated dimethylation at Lys20, acetylation at the N terminus, and acetylation at Lys16 (resolved from trimethylation, Deltam = 0.036 Da). A statistical evaluation reveals the critical role of mass accuracy and that PTM "isomers" are retrieved as next-best matches. The applicability of shotgun annotation to forms of H4 with up to six PTMs is demonstrated, with extensibility to other histones (e.g., H2A, H2B, H3) and other protein classes projected.

Targeted analysis and discovery of posttranslational modifications in proteins from methanogenic archaea by top-down MS.

For more complete characterization of DNA-predicted proteins (including their posttranslational modifications) a "top-down" approach using high-resolution tandem MS is forwarded here by its application to methanogens in both hypothesis-driven and discovery modes, with the latter dependent on new automation benchmarks for intact proteins. With proteins isolated from ribosomes and whole-cell lysates of Methanococcus jannaschii (approximately 1,800 genes) using a 2D protein fractionation method, 72 gene products were identified and characterized with 100% sequence coverage via automated fragmentation of intact protein ions in a custom quadrupole/Fourier transform hybrid mass spectrometer. Three incorrect start sites and two modifications were found, with one of each determined for MJ0556, a 20-kDa protein with an unknown methylation at approximately 50% occupancy in stationary phase cells. The separation approach combined with the quadrupole/Fourier transform hybrid mass spectrometer allowed targeted and efficient comparison of histones from M. jannaschii, Methanosarcina acetivorans (largest Archaeal genome, 5.8 Mb), and yeast. This finding revealed a striking difference in the posttranslational regulation of DNA packaging in Eukarya vs. the Archaea. This study illustrates a significant evolutionary step for the MS tools available for characterization of WT proteins from complex proteomes without proteolysis.

Web and database software for identification of intact proteins using "top down" mass spectrometry.

For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).