Accurate Protein Complex Retrieval by Affinity Enrichment Mass Spectrometry (AE-MS) Rather than Affinity Purification Mass Spectrometry (AP-MS)

Protein–protein interactions are fundamental to the understanding of biological processes. Affinity purification coupled to mass spectrometry (AP-MS) is one of the most promising methods for their investigation. Previously, complexes were purified as much as possible, frequently followed by identification of individual gel bands. However, todays mass spectrometers are highly sensitive, and powerful quantitative proteomics strategies are available to distinguish true interactors from background binders. Here we describe a high performance affinity enrichment-mass spectrometry method for investigating protein–protein interactions, in which no attempt at purifying complexes to homogeneity is made. Instead, we developed analysis methods that take advantage of specific enrichment of interactors in the context of a large amount of unspecific background binders. We perform single-step affinity enrichment of endogenously expressed GFP-tagged proteins and their interactors in budding yeast, followed by single-run, intensity-based label-free quantitative LC-MS/MS analysis. Each pull-down contains around 2000 background binders, which are reinterpreted from troubling contaminants to crucial elements in a novel data analysis strategy. First the background serves for accurate normalization. Second, interacting proteins are not identified by comparison to a single untagged control strain, but instead to the other tagged strains. Third, potential interactors are further validated by their intensity profiles across all samples. We demonstrate the power of our AE-MS method using several well-known and challenging yeast complexes of various abundances. AE-MS is not only highly efficient and robust, but also cost effective, broadly applicable, and can be performed in any laboratory with access to high-resolution mass spectrometers.Protein–protein interactions are key to protein-mediated biological processes and influence all aspects of life. Therefore, considerable efforts have been dedicated to the mapping of protein–protein interactions. A classical experimental approach consists of co-immunoprecipitation of protein complexes combined with SDS-PAGE followed by Western blotting to identify complex members. More recently, high-throughput techniques have been introduced; among these affinity purification-mass spectrometry (AP-MS)¹ (1–3) and the yeast two-hybrid (Y2H) approach (4–6) are the most prominent. AP-MS, in particular, has great potential for detecting functional interactions under near-physiological conditions, and has already been employed for interactome mapping in several organisms (7–15). Various AP-MS approaches have evolved over time, that differ in expression, tagging, and affinity purification of the bait protein; fractionation, LC-MS measurement, and quantification of the sample; and in data analysis. Recent progress in the AP-MS field has been driven by two factors: A new generation of mass spectrometers (16) providing higher sequencing speed, sensitivity, and mass accuracy, and the development of quantitative MS strategies.In the early days of AP-MS, tagged bait proteins were mostly overexpressed, enhancing their recovery in the pull-down. However, overexpression comes at the cost of obscuring the true situation in the cell, potentially leading to the detection of false interactions (17). Today, increased MS instrument power helps in the detection of bait proteins and interactors expressed at endogenous levels, augmenting the chances to detect functional interactions. In some simple organisms like yeast, genes of interest can directly be tagged in their genetic loci and expressed under their native promoter. In higher organisms, tagging proteins in their endogenous locus is more challenging, but also for mammalian cells, methods for close to endogenous expression are available. For instance, in controlled inducible expression systems, the concentration of the tagged bait protein can be titrated to close to endogenous levels (18). A very powerful approach is BAC transgenomics (19), as used in our QUBIC protocol (20), where a bacterial artificial chromosome (BAC) containing a tagged version of the gene of interest including all regulatory sequences and the natural promoter is stably transfected into a host cell line.The affinity purification step has also been subject to substantial changes over time. Previously, AP has been combined with nonquantitative MS as the readout, meaning all proteins identified by MS were considered potential interactors. Therefore, to reduce co-purifying “contaminants,” stringent two-step AP protocols using dual affinity tags like the TAP-tag (21) had to be employed. However, such stringent and multistep protocols can result in the loss of weak or transient interactors (3), whereas laborious and partially subjective filtering still has to be applied to clean up the list of identified proteins. The introduction of quantitative mass spectrometry (22–25) to the interactomics field about ten years ago was a paradigm shift, as it offered a proper way of dealing with unspecific binding and true interactors could be directly distinguished from background binders (26, 27). Importantly, quantification enables the detection of true interactors even under low-stringent conditions (28). In turn, this allowed the return to single-step AP protocols, which are milder and faster, and hence more suitable for detecting weak and transient interactors.Despite these advances, nonquantitative methods—often in combination with the TAP-tagging approach—are still popular and widely used, presumably because of reagent expenses and labeling protocols used in label-based approaches. However, there are ways to determine relative protein abundances in a label-free format. A simple, semiquantitative label-free way to estimate protein abundance is spectral counting (29). Another relative label-free quantification strategy is based on peptide intensities (30). In recent years high resolution MS has become much more widely accessible and there has been great progress in intensity-based label-free quantification (LFQ) approaches. Together with development of sophisticated LFQ algorithms, this has boosted obtainable accuracy. Intensity-based LFQ now offers a viable and cost-effective alternative to label-based methods in most applications (31). The potential of intensity-based LFQ approaches as tools for investigating protein–protein interactions has already been demonstrated by us (20, 32, 33) and others (34, 35). We have further refined intensity-based LFQ in the context of the MaxQuant framework (36) using sophisticated normalization algorithms, achieving excellent accuracy and robustness of the measured “MaxLFQ” intensities (37).Another important advance in AP-MS, again enabled by increased MS instrument power, was the development of single-shot LC-MS methods with comprehensive coverage. Instead of extensive fractionation, which was previously needed to reduce sample complexity, nowadays even entire model proteomes can be measured in single LC-MS runs (38). The protein mixture resulting from pull-downs is naturally of lower complexity compared with the entire proteome. Therefore, modern MS obviates the need for gel-based (or other) fractionation and samples can be analyzed in single runs. Apart from avoiding selection of gel bands by visual examination, this has many advantages, including decreased sample preparation and measurement time, increased sensitivity, and higher quantitative accuracy in a label-free format.In this work, we build on many of the recent advances in the field to establish a state of the art LFQ AE-MS method. Based on our previous QUBIC pipeline (20), we developed an approach for investigating protein–protein interactions, which we exemplify in Saccharomyces cerevisiae. We extended the data analysis pipeline to extract the wealth of information contained in the LFQ data, by establishing a novel concept that specifically makes use of the signature of background binders instead of eliminating them from the data set. The large amount of unspecific binders detected in our experiments rendered the use of a classic untagged control strain unnecessary and enabled comparing to a control group consisting of many unrelated pull-downs instead. Our protocol is generic, practical, and fast, uses low input amounts, and identifies interactors with high confidence. We propose that single-step pull-down experiments, especially when coupled to high-sensitivity MS, should now be regarded as affinity enrichment rather than affinity purification methods.     

Sharing natural resources: mountain gorillas and people in the Parc National des Volcans,Rwanda

The compatibility of natural resource use by people and mountain gorillas (Gorilla beringei beringei) within the Parc National des Volcans was studied. The distribution of gorillas was modelled using a Maximum Entropy algorithm. Biophysical predictor variables were trained with daily GPS locations of gorillas during 2006. Elevation, as a climate surrogate, was the best predictor (58%) of the occurrence of gorillas. The mid‐altitudes (2500–3500 m a.s.l.) contained the bulk of the gorilla groups. Incoming solar radiation, as proxy for comfortable nesting sites, was the second best predictor (17%). Vegetation types, as foliage provider, (13%) and slope steepness for providing security (12%) were contributing predictors. The modelled and actual gorilla distributions were together overlaid with people’s resource use in the park. Both people and gorillas were congregated in the areas identified as most suitable for gorillas. However, within these areas spatial segregation was found between human natural resource‐users and gorillas. Therefore, the number of gorillas is likely to be limited by the human natural resource use within the park. A perimeter fence, the introduction of community‐based natural resource management, and a buffer zone are discussed as short‐, medium‐ and long‐term mitigation measures.     

Neuroprotective properties of levosimendan in an in vitro model of traumatic brain injury

Background  

We investigated the neuroprotective properties of levosimendan, a novel inodilator, in an in vitro model of traumatic brain injury.     

Matrix metalloproteinases in the cervical mucus plug in relation to gestational age,plug compartment,and preterm labor

Background  

High concentrations of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been identified in the cervical mucus plug (CMP) at term of pregnancy. Their physiological and pathophysiological implications, however, remain to be elucidated, and CMPs from preterm labor have never been examined. This study was therefore conducted to describe the concentrations of MMP-2, TIMP-1, MMP-8 and MMP-9 in the CMP in relation to gestational age, IL-8 as an indicator of inflammation, compartment of the CMP, and preterm labor.     

Application of Crustacean Chitin as a Co-diluent in Direct Compression of Tablets

A “simplex-centroid mixture design” was used to study the direct-compression properties of binary and ternary mixtures of chitin and two cellulosic direct-compression diluents. Native milled and fractioned (125–250 μm) crustacean chitin of lobster origin was blended with microcrystalline cellulose, MCC (Avicel® PH 102) and spray-dried lactose–cellulose, SDLC Cellactose® (composed of a spray-dried mixture of alpha-lactose monohydrate 75% and cellulose powder 25%). An instrumented single-punch tablet machine was used for tablet compactions. The flowability of the powder mixtures composed of a high percentage of chitin and SDLC was clearly improved. The fractioned pure chitin powder was easily compressed into tablets by using a magnesium stearate level of 0.1% (w/w) but, as the die lubricant level was 0.5% (w/w), the tablet strength collapsed dramatically. The tablets compressed from the binary mixtures of MCC and SDLC exhibited elevated mechanical strengths (>100 N) independent of the die lubricant level applied. In conclusion, fractioned chitin of crustacean origin can be used as an abundant direct-compression co-diluent with the established cellulosic excipients to modify the mechanical strength and, consequently, the disintegration of the tablets. Chitin of crustacean origin, however, is a lubrication-sensitive material, and this should be taken into account in formulating direct-compression tablets of it.     

Characterization of immune responses against gastrointestinal nematodes in weaned lambs grazing willow fodder blocks

A 131-day rotational grazing experiment was conducted in the summer autumn of 2007/2008 to compare effects of feeding condensed tannin (CT)-containing willow (Salix spp.) fodder blocks (i.e., silvopastoral system) or perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) control pasture upon the immune response to gastrointestinal nematode parasite infection in Romney weaned lambs. Groups of lambs (n = 40) were allocated to either willow fodder blocks or control pasture; half of each group (n = 20) were regularly-drenched with anthelmintic at approximately 21 day intervals, whilst the remaining 20 weaned lambs were not drenched unless pre-determined faecal nematode egg counts (FEC) were reached, when all weaned lambs in that group were drenched with anthelmintic (i.e., trigger-drenching). Metabolizable energy and CT concentrations were higher in willow fodder versus pasture herbages. Weaned lambs grazing willow fodder blocks had lower live weight gain (92 g/day) and carcass weight (14.4 kg) than those grazing control pasture (134 g; 15.3 kg), with no effect of anthelmintic drenching. Regular anthelmintic treatment maintained similar and low FEC up to day 82, which then increased, whilst trigger-drenched lambs grazing willow fodder blocks had higher FEC than lambs grazing control pasture on three out of eight occasions. As judged by faecal larval cultures, grazing willow fodder blocks reduced the relative proportions of abomasal-dwelling parasites (Haemonchus contortus and Teladorsagia spp.). Trigger-drenched willow fodder block-fed sheep had higher platelet, eosinophil, total white blood cell and lymphocyte counts, greater CD21⁺ and greater γδ (Gamma Delta) TCR⁺ (T cell receptor) lymphocyte subsets than control pasture-fed sheep, and higher plasma levels of Immunoglobulin A (IgA) specific for carbohydrate larval antigen (CarLa) on day 105 (P<0.001). None of these parameters were affected by grazing treatment in regularly-drenched lambs. Higher immunological measurements in trigger-drenched lambs grazing willow fodder blocks could be due to higher larval intake and/or to the effects of secondary compounds in willow fodder blocks priming the immune system. Further research is required to separate these effects.