European Turtle Dove Streptopelia turtur diet composition in Southern Spain: the role of wild seeds in Mediterranean forest areas

Capsule: The diet of European Turtle Doves Streptopelia turtur in Mediterranean forest contained a large volume of wild plant seed but from a small number of key species.

Aims: To determine which seed species are consumed by Turtle Doves in Mediterranean forest areas.

Methods: Digestive tract contents were identified and evaluated for 222 Turtle Doves shot by hunters during three consecutive years.

Results: Thirty seed species were identified in the diet, but only a few species represented most of the volume and frequency. Wild plant seeds appeared in 65.8% of digestive tracts and showed significant variation between years. Although the main wild seed species consumed each year varied annually, certain species were found in the diet every year in high volume and frequency. Adults showed a more diverse and numerous consumption of wild seeds than did juveniles. Plastic granules were also found in 3.8% of individuals.

Conclusion: A greater number of wild seed species was found in the diet in contrast to previous studies performed in farmland. Echium plantagineum and Amaranthus deflexus could be important seed sources for Turtle Doves in Mediterranean forest. Additionally, the herbaceous species found in the diet whose seeds ripen earlier in the season might play an important role in Turtle Dove reproductive performance, since they are frequently the only available food in the first half of the breeding season.     

Early detection of bacterial diseases in apple plants by analysis of volatile organic compounds profiles and use of electronic nose

DNA‐based protocols are the standard methods for the diagnosis of infected plant material. Nevertheless, these methods are time‐consuming and require trained personnel, with an efficacy depending on the sampling procedure. In comparison, recognition methods based on volatile compounds emissions are less precise, but allow a non‐destructive mass screening of bulk samples, and may be implemented to steer molecular diagnosis. In this study, the analysis of volatile compounds is used for the discrimination of fire blight (Erwinia amylovora) and blossom blight (Pseudomonas syringae pv. syringae) on apple propagation material. Possible marker compounds were identified by gas chromatography–mass spectroscopy (GC‐MS) and proton transfer reaction‐time of flight‐mass spectroscopy (PTR‐ToF‐MS). In addition, two commercial electronic noses were used for diagnosis. After a preliminary validation in vitro, a diagnostic protocol was successfully developed to scale up to real nursery conditions on cold stored, asymptomatic dormant plants.     

Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling

We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754).Proteins rarely work as monomers to carry out all the biological processes needed for cells to function. An estimate of the total number of protein-protein interactions within the human proteome, based on currently available data sets, is ∼650,000 (1). This is likely an underestimate, given that many proteins form either transient, or weak interactions within intact cells that may not yet have been detected. This suggests that the majority of human proteins can participate in protein complex formation, at least under some conditions. This includes the many well-studied soluble protein complexes in the cytoplasm, exemplified by the proteasome, ribosomes and cytoskeletal network. It also includes many membrane-associated complexes, for example receptor tyrosine kinase signaling complexes, integrin networks and transmembrane transporters (2). To characterize the many roles of multi-protein complexes in biological regulatory mechanisms, it is important to have convenient methods for the rapid and efficient analysis of their composition and dynamics (3). Ideally, such methods should be applicable to system-wide studies and allow the analysis of endogenous proteins, rather than exclusively use tagged and/or over-expressed baits.The methods available for the proteome-wide analysis of protein interactions have developed swiftly over the last ten years. This field is dominated by affinity-enrichment based approaches, using either tagged constructs, or antibodies specific for endogenous proteins. Another approach is in vivo proximity labeling, based, for example, on the exogenous expression of a protein of interest, fused either to a promiscuous biotin-ligase (BioID) (4), or to a peroxidase enzyme that activates biotin-phenol (APEX) (5). While these data sets have proved very useful, there are some downsides. For example, a large expense in terms of both time and money to generate the thousands of individual “bait” proteins required for global interaction analyses. In addition, each of these affinity enrichments will be performed in only one type of buffer system, which is unlikely to be compatible with the maintenance of all protein-protein interactions. Another dimension to the analytical problem is that many proteins are expressed as different sized isoforms and/or in different post-translationally modified forms, resulting in formation of multiple, related, but functionally distinct complexes, with different combinations of interaction partners (6). Using affinity-enrichment/pull-down methods alone makes it difficult to resolve such mixtures of different forms of related protein complexes, complicating a detailed understanding of biological response mechanisms.An alternative strategy involves protein correlation profiling-MS, i.e. correlating similarities in the fractionation profiles of proteins detected by mass spectrometry, assuming that proteins in a common complex will cofractionate. This approach was previously applied to the analysis of subcellular organelle proteomes (7, 8), and subsequently extended to analyze soluble protein complexes. Thus, recent studies have shown that chromatography-based separation of soluble protein complexes, combined with fraction collection and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS)¹, facilitates analysis of many hundreds of soluble complexes from a single experiment (6, 9–11). A limitation of all of these studies, however, is that the native extraction conditions used to preserve protein-protein interactions isolates predominantly stable, soluble complexes. For example, many proteins that are integral to membranes are not recovered (12). Similarly, soluble protein complexes that have weakly bound protein subunits can dissociate upon cell lysis and the inevitable dilution associated with extraction. Thus, the potential value of this approach for the system-wide analysis of protein complexes is limited without a covalent tether to hold protein-protein interactions intact during extraction and subsequent chromatographic separation (13).Covalent protein crosslinking has been used extensively to stabilize protein complexes, cultured cells and tissues for subsequent analysis, either by microscopy, nucleotide sequencing or mass spectrometry. The agents employed to crosslink proteins to each other include various chemical groups able to react with the side-chains of either amino acids, nucleotides, carbohydrates or lipids (14). These crosslinking agents vary in the efficiency with which they perfuse into unbroken cells/tissues and the speed of their reaction when in proximity to a suitable chemical group. One of the most widely used crosslinkers is formaldehyde, which can reversibly form a covalent crosslink to stabilize both protein-protein and protein-nucleotide interactions (15–21). One of the main benefits of using formaldehyde is that because of its small size, it readily permeates intact cells and tissues. Another benefit of using formaldehyde is the easy reversal of the crosslinks by heating and subsequent compatibility with mass spectrometry-based proteome analysis.Here, we describe a mass spectrometry-based proteomic approach for the efficient global analysis of protein complexes, including membrane proteins, using in vivo protein crosslinking combined with denaturing extraction. Using high-resolution, size-exclusion chromatography (SEC) to separate crosslinked complexes under denaturing conditions and MS analysis of fractionated proteins, we could identify membrane bound and membrane associated complexes not accessible in native extracts. We present a detailed comparison of the sets of protein complexes that can be identified using protein correlation profiling MS analysis in conjunction with both formaldehyde crosslinked and native extracts from U2OS cells. We provide access to the entire proteome-wide data sets of both in vivo crosslinked and native U2OScell protein complexes via a searchable online database (http://www.peptracker.com/epd/).