Applications of diagonal chromatography for proteome-wide characterization of protein modifications and activity-based analyses

Numerous gel-free proteomics techniques have been reported over the past few years, introducing a move from proteins to peptides as bits of information in qualitative and quantitative proteome studies. Many shotgun proteomics techniques randomly sample thousands of peptides in a qualitative and quantitative manner but overlook the vast majority of protein modifications that are often crucial for proper protein structure and function. Peptide-based proteomic approaches have thus been developed to profile a diverse set of modifications including, but not at all limited, to phosphorylation, glycosylation and ubiquitination. Typical here is that each modification needs a specific, tailor-made analytical procedure. In this minireview, we discuss how one technique - diagonal reverse-phase chromatography - is applied to study two different types of protein modification: protein processing and protein N-glycosylation. Additionally, we discuss an activity-based proteome study in which purine-binding proteins were profiled by diagonal chromatography.     

A la carte proteomics with an emphasis on gel-free techniques

Since the introduction of the proteome term somewhat more than a decade ago the field of proteomics witnessed a rapid growth mainly fueled by instrumental analytical improvements. Of particular notice is the advent of a diverse set of gel-free proteomics techniques. In this review, we discuss several of these gel-free techniques both for monitoring protein concentration changes and protein modifications, in particular protein phosphorylation, glycosylation, and protein processing. Furthermore, different approaches for (multiplexed) gel-free proteome analysis are discussed.     

Light and warming drive forest understorey community development in different environments

Plant community composition and functional traits respond to chronic drivers such as climate change and nitrogen (N) deposition. In contrast, pulse disturbances from ecosystem management can additionally change resources and conditions. Community responses to combined environmental changes may further depend on land‐use legacies. Disentangling the relative importance of these global change drivers is necessary to improve predictions of future plant communities. We performed a multifactor global change experiment to disentangle drivers of herbaceous plant community trajectories in a temperate deciduous forest. Communities of five species, assembled from a pool of 15 forest herb species with varying ecological strategies, were grown in 384 mesocosms on soils from ancient forest (forested at least since 1850) and postagricultural forest (forested since 1950) collected across Europe. Mesocosms were exposed to two‐level full‐factorial treatments of warming, light addition (representing changing forest management) and N enrichment. We measured plant height, specific leaf area (SLA) and species cover over the course of three growing seasons. Increasing light availability followed by warming reordered the species towards a taller herb community, with limited effects of N enrichment or the forest land‐use history. Two‐way interactions between treatments and incorporating intraspecific trait variation (ITV) did not yield additional inference on community height change. Contrastingly, community SLA differed when considering ITV along with species reordering, which highlights ITV’s importance for understanding leaf morphology responses to nutrient enrichment in dark conditions. Contrary to our expectations, we found limited evidence of land‐use legacies affecting community responses to environmental changes, perhaps because dispersal limitation was removed in the experimental design. These findings can improve predictions of community functional trait responses to global changes by acknowledging ITV, and subtle changes in light availability. Adaptive forest management to impending global change could benefit the restoration and conservation of understorey plant communities by reducing the light availability.     

Seed banks of temperate deciduous forests during secondary succession

Question: (i) How does former land use and land use intensity affect seed bank development during post‐agricultural succession? (ii) How does time since the last clear‐cut change seed bank composition during post‐clear‐cut succession? Methods: One data set was compiled per succession type using the following selection criteria: (i) the data set included a successional series, (ii) plots were located in mesotrophic forest plant communities and (iii) vegetation data were available. The post‐agricultural succession data set comprised 76 recent forest plots (eight studies); the post‐clear‐cut succession data set comprised 218 ancient forest plots (three studies). Each data set was analysed separately using either linear mixed models or generalized linear models, controlling for both environmental heterogeneity and variation between study locations. Results: In the post‐agricultural succession data set, land use and time significantly affected nearly all the studied seed bank characteristics. Seed banks on former arable land recovered poorly even after 150 year of restored forest cover, whereas moderate land use intensities (grasslands, heathlands) yielded more rapid seed bank recovery. Time was a significant determinant of all but two soil seed bank characteristics during post‐clear‐cut succession. Seed banks in managed ancient forest differed strongly in their characteristics compared to primary forest seed banks. Conclusions: Forest seed banks bear the marks of former land use and/or forest management and continue to do so for at least 150 years. Nevertheless, time since the last major disturbance, being either former land use or clear‐cutting, remains a significant determinant of the seed bank.     

Comparison of three assays for the quantification of Candida biomass in suspension and CDC reactor grown biofilms

A common assay to measure yeast metabolic activity in biofilms is based on the reduction of the tetrazolium salt XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} to a colored formazan. However, a recent report, also confirmed by our own findings about the shortcomings of the chromogenic XTT assay, has prompted us to investigate alternative methods for yeast biomass quantification. To this end, two fluorogenic assays using fluorescein diacetate (FDA) and SYTO 9 as well as the XTT assay were comparatively evaluated with regard to the linear range of Candida albicans and Candida parapsilosis cell number-response curves, precision and intra- and interspecies variability. Reading of fluorescence and absorbance was carried out in a multilabel microtiter plate reader. All three assays were adequate for the determination of planktonic yeast biomass, but the FDA and SYTO 9 assays present practical advantages. When applied to the quantification of yeast biofilm biomass obtained in the CDC biofilm reactor, the FDA assay proved superior.     

Detailed kinetics of the direct allo-response in human liver transplant recipients: new insights from an optimized assay

Conventional assays for quantification of allo-reactive T-cell precursor frequencies (PF) are relatively insensitive. We present a robust assay for quantification of PF of T-cells with direct donor-specificity, and establish the kinetics of circulating donor-specific T cells after liver transplantation (LTx). B cells from donor splenocytes were differentiated into professional antigen-presenting cells by CD40-engagement (CD40-B cells). CFSE-labelled PBMC from LTx-recipients obtained before and at several time points after LTx, were stimulated with donor-derived or 3^rd party CD40-B cells. PF of donor-specific T cells were calculated from CFSE-dilution patterns, and intracellular IFN-γ was determined after re-stimulation with CD40-B cells. Compared to splenocytes, stimulations with CD40-B cells resulted in 3 to 5-fold higher responding T-cell PF. Memory and naïve T-cell subsets responded equally to allogeneic CD40-B cell stimulation. Donor-specific CD4⁺ and CD8⁺ T-cell PF ranged from 0.5 to 19% (median: 5.2%). One week after LTx, PF of circulating donor-specific CD4⁺ and CD8⁺ T cells increased significantly, while only a minor increase in numbers of T cells reacting to 3^rd party allo-antigens was observed. One year after LTx numbers of CD4⁺ and CD8⁺ T cells reacting to donor antigens, as well as those reacting to 3^rd party allo-antigens, were slightly lower compared to pre-transplant values. Moreover, CD4⁺ and CD8⁺ T cells responding to donor-derived, as well as those reacting to 3^rd party CD40-B cells, produced less IFN-γ. In conclusion, our alternative approach enables detection of allo-reactive human T cells at high frequencies, and after application we conclude that donor-specific T-cell PF increase immediately after LTx. However, no evidence for a specific loss of circulating T-cells recognizing donor allo-antigens via the direct pathway up to 1 year after LTx was obtained, underscoring the relative insensitiveness of previous assays.     

Construction of a two-dimensional gel electrophoresis protein database for the Nicotiana tabacum cv. Bright Yellow-2 cell suspension culture

Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2). The BY-2 cell suspension culture is widely used as a model system to study the growth and development of plant cells. We present a protocol describing the sample preparation and 2-DE, enabling us to separate and display more than 1000 proteins from this cell culture. A reference gel was generated, using immobilized pH gradient isoelectric focusing in a linear gradient from pH 3 to 10 and 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the tobacco genome is not sequenced yet, a range of protein spots from this reference map was identified by means of a semi-automated liquid chromatography-ESI-quadrupole time of flight-tandem MS (LC-ESI-QTOF-MS-MS) setup and cross-species matching. These data were integrated in a database, which can be accessed at http://tby2-www.uia.ac.be/tby2/. On the on-line reference map, the identified protein spots are hyperlinked to individual protein entries. Each protein entry contains all identification information, as well as links to relevant entries in other on-line databases. Comprehensive search functions are implemented. Especially for an unsequenced but widespread model organism like tobacco BY-2, such a reference database is a convenient source for protein information that brings protein identification within reach without the need for extensive MS. This publicly accessible database provides a solid basis for tobacco BY-2 proteomics in the future.     

Loss of guanylyl cyclase C (GCC) signaling leads to dysfunctional intestinal barrier

Background

Guanylyl Cyclase C (GCC) signaling via uroguanylin (UGN) and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function.

Methodology/Principal Findings

Paracellular permeability of intestinal segments was assessed in wild type (WT) and GCC deficient (GCC−/−) mice with and without lipopolysaccharide (LPS) challenge, as well as in UGN deficient (UGN−/−) mice. IFNγ and myosin light chain kinase (MLCK) levels were determined by real time PCR. Expression of tight junction proteins (TJPs), phosphorylation of myosin II regulatory light chain (MLC), and STAT1 activation were examined in intestinal epithelial cells (IECs) and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi). We found that intestinal permeability was increased in GCC−/− and UGN−/− mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC−/− mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC−/− and UGN−/− mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability.

Conclusions/Significance

GCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury.     

Error-proneness as a handicap signal

This paper describes two discrete signalling models in which the error-proneness of signals can serve as a handicap signal. In the first model, the direct handicap of sending a high-quality signal is not large enough to assure that a low-quality signaller will not send it. However, if the receiver sometimes mistakes a high-quality signal for a low-quality one, then there is an indirect handicap to sending a high-quality signal. The total handicap of sending such a signal may then still be such that a low-quality signaller would not want to send it. In the second model, there is no direct handicap of sending signals, so that nothing would seem to stop a signaller from always sending a high-quality signal. However, the receiver sometimes fails to detect signals, and this causes an indirect handicap of sending a high-quality signal that still stops the low-quality signaller of sending such a signal. The conditions for honesty are that the probability of an error of detection is higher for a high-quality than for a low-quality signal, and that the signaller who does not detect a signal adopts a response that is bad to the signaller. In both our models, we thus obtain the result that signal accuracy should not lie above a certain level in order for honest signalling to be possible. Moreover, we show that the maximal accuracy that can be achieved is higher the lower the degree of conflict between signaller and receiver. As well, we show that it is the conditions for honest signalling that may be constraining signal accuracy, rather than the signaller trying to make honest signals as effective as possible given receiver psychology, or the signaller adapting the accuracy of honest signals depending on his interests.