Coastal marine ecosystems are highly vulnerable to anthropogenically introduced invasive species, in part because movement of organisms between diverse and widely separated habitats can occur rapidly. High rates of translocation, most notably via the ballast water of ocean-going ships (Carlton and Geller, 1993), allow potential invaders repeated opportunities to colonize coastal habitats. Indeed, the result of such continual introductions over the past century has been a rapid increase in the number of successful colonizations by coastal and estuarine species (Cohen and Carlton, 1998). Clearly, however, not all species introduced into novel habitats colonize successfully, and a number of generalizations have been put forward to explain why certain invaders are able to compete successfully against aboriginal species (Kolar and Lodge, 2001;Lodge, 1993). These include high dispersal rates, phenotypic plasticity and pre-existing physiological suitability for the invaded habitat. Here, we explore whether biochemical adaptation to temperature potentially may play a role in invasion success, by comparing temperature sensitivity of the enzyme cytosolic malate dehydrogenase (cMDH) from three mussel congeners. Two of these mussels, Mytilus trossulus and M. californianus, are native to the west coast of North America and the third, M. galloprovincialis, is a species introduced there from the Mediterranean Sea.Mytilus trossulus and M. galloprovincialis are members of To test the effects of individual substitutions on kinetic properties, we used site-directed mutagenesis to create recombinant cMDHs. Recombinant wild-type M. trossulus cMDH (rWT) has high K m NADH compared with mutants incorporating the non-conservative substitutions found in M. californianus and M. galloprovincialis -V114H and V114N, respectively -demonstrating that these mutations are responsible for the differences found in substrate affinity. Turnover number (k cat ) is also higher in rWT compared with the two mutants, consistent with cold adaptation in the M. trossulus ortholog. Conversely, rWT and V114H appear more thermostable than V114N. Based on a comparison of K m NADH and k cat values among the orthologs, we propose that immersion temperatures are of greater selective importance in adapting kinetic properties than the more extreme temperatures that occur during emersion. The relative warm adaptation of M. galloprovincialis cMDH may be one of a suite of physiological characters that enhance the competitive ability of this invasive species in warm habitats.
The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-
The ability to perform thorough sampling is of critical importance when using mass spectrometry to characterize complex proteomic mixtures. A common approach is to re-interrogate a sample multiple times by LC-MS/MS. However, the conventional data-dependent acquisition methods that are typically used in proteomics studies will often redundantly sample high-intensity precursor ions while failing to sample low-intensity precursors entirely. We describe a method wherein the masses of successfully identified peptides are used to generate an accurate mass exclusion list such that those precursors are not selected for sequencing during subsequent analyses. We performed multiple concatenated analytical runs to sample a complex cell lysate, using either accurate mass exclusionbased data-dependent acquisition (AMEx) or standard data-dependent acquisition, and found that utilization of AMEx on an ESI-Orbitrap instrument significantly increases the total number of validated peptide identifications relative to a standard DDA approach. The additional identified peptides represent precursor ions that exhibit low signal intensity in the sample. Increasing the total number of peptide identifications augmented the number of proteins identified, as well as improved the sequence coverage of those proteins. Together, these data indicate that using AMEx is an effective strategy to improve the characterization of complex proteomic mixtures.
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