Basin-scale distribution patterns of photosynthetic picoeukaryotes along an Atlantic Meridional Transect

Photosynthetic picoeukaryotes (PPEs) of a size < 3 μm play a crucial role in oceanic primary production. However, little is known of the structure of the PPE community over large spatial scales. Here, we investigated the distribution of various PPE classes along an Atlantic Meridional Transect sampled in boreal autumn 2004 that encompasses a range of ocean provinces (gyres, upwelling, temperate regions), using dot blot hybridization technology targeting plastid 16S rRNA gene amplicons. Two algal classes, Prymnesiophyceae and Chrysophyceae, dominated the PPE community throughout the Atlantic Ocean, over a range of water masses presenting different trophic profiles. However, these classes showed strongly complementary distributions with Chrysophyceae dominating northern temperate waters, the southern gyre and equatorial regions, while prymnesiophytes dominated the northern gyre. Phylogenetic analyses using both plastid and nuclear rRNA genes revealed a high diversity among members of both classes, including sequences contained in lineages with no close cultured counterpart. Other PPE classes were less prevalent along the transect, with members of the Cryptophyceae, Pelagophyceae and Eustigmatophyceae essentially restricted to specific regions. Multivariate statistical analyses revealed strong relationships between the distribution patterns of some of these latter PPE classes and temperature, light intensity and nutrient concentrations. Cryptophyceae, for example, were mostly found in the upwelling region and associated with higher nutrient concentrations. However, the key classes of Prymnesiophyceae and Chrysophyceae were not strongly influenced by the variables measured. Although there appeared to be a positive relationship between Chrysophyceae distribution and light intensity, the complementary distributions of these classes could not be explained by the variables recorded and this requires further explanation.     

Schistosoma comparative genomics: integrating genome structure, parasite biology and anthelmintic discovery

Schistosoma genomes provide a comprehensive resource for identifying the molecular processes that shape parasite evolution and for discovering novel chemotherapeutic or immunoprophylactic targets. Here, we demonstrate how intragenus and intergenus comparative genomics can be used to drive these investigations forward, illustrate the advantages and limitations of these approaches and review how post-genomic technologies offer complementary strategies for genome characterisation. Although sequencing and functional characterisation of other schistosome/platyhelminth genomes continues to expedite anthelmintic discovery, we contend that future priorities should equally focus on improving assembly quality, and chromosomal assignment, of existing schistosome/platyhelminth genomes.     

Expression and shedding of endothelial protein C receptor in prostate cancer cells

Background  

Increasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines.     

Universal and confident phosphorylation site localization using phosphoRS

An algorithm for the assignment of phosphorylation sites in peptides is described. The program uses tandem mass spectrometry data in conjunction with the respective peptide sequences to calculate site probabilities for all potential phosphorylation sites. Tandem mass spectra from synthetic phosphopeptides were used for optimization of the scoring parameters employing all commonly used fragmentation techniques. Calculation of probabilities was adapted to the different fragmentation methods and to the maximum mass deviation of the analysis. The software includes a novel approach to peak extraction, required for matching experimental data to the theoretical values of all isoforms, by defining individual peak depths for the different regions of the tandem mass spectrum. Mixtures of synthetic phosphopeptides were used to validate the program by calculation of its false localization rate versus site probability cutoff characteristic. Notably, the empirical obtained precision was higher than indicated by the applied probability cutoff. In addition, the performance of the algorithm was compared to existing approaches to site localization such as Ascore. In order to assess the practical applicability of the algorithm to large data sets, phosphopeptides from a biological sample were analyzed, localizing more than 3000 nonredundant phosphorylation sites. Finally, the results obtained for the different fragmentation methods and localization tools were compared and discussed.     

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.The evolving maturation of the field of proteomics has, in the same way as in genomics, highlighted the need of better sampling procedures and sample preparation methodologies to minimize the effect of post mortem alterations. The aspect of sample quality is not new in any way and is relevant in most biomedical fields but has only lately started to receive adequate attention. The main factors influencing sample quality is storage temperature of the body until tissue removal (foremost a problem in clinical settings and extraction of less accessible tissue samples from model organisms) and post mortem interval (PMI)¹ (1–3). Post mortem degradation in during PMI is a well known compromising problem when studying endogenous peptides (2, 3) and has also been proven to affect the results of polypeptide (here defined as proteins larger than 10 kDa) studies (3–8). PMI degradation has mainly been studied on human or mouse brain tissue, using two-dimensional electrophoresis (2-DE), SDS-PAGE, and immunoblotting (1, 3–12). There are also a few proteomic studies on muscle tissue degradation in livestock (13–16).We and others have previously explored the effect of focused microwave irradiation with regard to sample quality, demonstrating that this method is more reliable than snap freezing in liquid nitrogen, especially with regard to post-translational modification (PTM) stability (2, 3, 17–20). An alternative method based on cryostat dissection with subsequent heat treatment through boiling has also been reported to improve endogenous peptide sample quality (21). Besides focused microwave irradiation, which is specifically used for rodent brain tissue sampling, we have also demonstrated the efficiency of rapid heat stabilization through conductivity with regard to sample degradation (3, 22). Although somewhat constrained by its dependence on how quickly the tissue is harvested from the body, the latter procedure has the added advantage that it can be used on any type of tissue and species, fresh as well as frozen. This study will compare effects of sampling procedures on the liver and pancreas degradome following rapid heat stabilization, the more traditional snap freezing, or the combination of snap freezing with subsequent heat stabilization.To summarize, this study investigated the effects of post mortem degradation in pancreas and liver. Both tissues are well studied because of their multiple functions in the body and their involvement in different diseases such as diabetes or hepatocarcinoma. Pancreas is especially interesting in this context as it displays endocrine secretion of peptides, and exocrine secretion of digestive enzymes, the later making it a protease rich tissue. We used both two-dimensional difference in gel electrophoresis (2D-DIGE) and label free liquid chromatography mass spectrometry (LC-MS) based differential peptide display (2, 18), the later to better investigate changes in small molecular fragment that are not easily detectable by gel-based methods. 2D-DIGE is an unrivaled methodology to characterize alterations in isoform patterns, which is an important aspect considering that post-translational modifications (PTMs) such as phosphorylations are especially sensitive to post mortem influence within a few minutes PMI (3). The peptidomics approach has been used in several studies to point out early post mortem changes and protein degradation that tissue undergo following sampling and is therefore a well-suited method (3, 18, 22).     

Pressurized Pepsin Digestion in Proteomics: AN AUTOMATABLE ALTERNATIVE TO TRYPSIN FOR INTEGRATED TOP-DOWN BOTTOM-UP PROTEOMICS*

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.In-depth characterization and quantitation of protein isoforms, including post-translationally modified proteins, are challenging goals of contemporary proteomics. Traditionally, top-down (1, 2) and bottom-up (3, 4) proteomics have been two distinct analytical paths for liquid-based proteomics analysis. Top-down proteomics is the mass spectrometry (MS)-based characterization of intact proteins, whereas bottom-up proteomics requires a chemical or enzymatic proteolytic digestion of all proteins into peptides prior to MS analysis. Both strategies have their own strengths and challenges and can be thought of as complementary rather than competing analytical techniques.In a top-down proteomics approach, proteins are usually separated by one- or two-dimensional liquid chromatography (LC) and identified using high performance MS (5, 6). This approach is very attractive because it allows the identification of protein isoforms arising from various amino acid modifications, genetic variants (e.g. single nucleotide polymorphisms), mRNA splice variants, and multisite modifications (7) (e.g. specific histone modifications) as well as characterization of proteolytic processing events. However, there are several challenges that have limited the broad application of the approach. Typically, intact proteins are less soluble than their peptide complement, which effectively results in greater losses during various stages of sample handling (i.e. limited sensitivity). Similarly, proteins above ∼40–50 kDa in size are more difficult to ionize, detect, and dissociate in most high throughput MS work flows. Additionally, major challenges associated with MS data interpretation and sensitivity, especially for higher molecular mass proteins (>100 kDa) and highly hydrophobic proteins (e.g. integral membrane proteins), remain largely unsolved, thus limiting the applicability of top-down proteomics on a large scale.Bottom-up proteomics approaches have broad application because peptides are easier to separate and analyze via LC coupled with tandem mass spectrometry (MS/MS), offering a basis for more comprehensive protein identification. As this method relies on protein digestion (which produces multiple peptides for each protein), the sample complexity can become exceedingly large, requiring several dimensions of chromatographic separations (e.g. strong cation exchange and/or high pH reversed phase) prior to the final LC separation (typically reversed phase (RP)¹ C₁₈), which is oftentimes directly coupled with the mass spectrometer (3, 8). In general, the bottom-up analysis rarely achieves 100% sequence coverage of the original proteins, which can result in an incorrect/incomplete assessment of protein isoforms and combinatorial PTMs. Additionally, the digested peptides are not detected with uniform efficiency, which challenges and distorts protein quantification efforts.Because the data obtained from top-down and bottom-up work flows are complementary, several attempts have been made to integrate the two strategies (9, 10). Typically, these efforts have utilized extensive fractionation of the intact protein separation followed by bottom-up analysis of the collected fractions. Results so far have encouraged us to consider on-line digestion methods for integrating top-down and bottom-up proteomics in a higher throughput fashion. Such an on-line digestion approach would not only benefit in terms of higher sample throughput and improved overall sensitivity but would also allow a better correlation between the observed intact protein and its peptide digestion products, greatly aiding data analysis and protein characterization efforts.So far, however, none of the on-line integrated methods have proven robust enough for routine high throughput analyses. One of the reasons for this limited success relates to the choice of the proteolytic enzyme used for the bottom-up segment. Trypsin is by far the most widely used enzyme for proteome analyses because it is affordable (relative to other proteases), it has been well characterized for proteome research, and it offers a nice array of detectable peptides due to a fairly even distribution of lysines and arginines across most proteins. However, protein/peptide RPLC separations (optimal at low pH) are fundamentally incompatible with on-line trypsin digestion (optimal at pH ∼ 8) (11, 12). Therefore, on-line coupling of trypsin digestion and RPLC separations is fraught with technological challenges, and proposed solutions (12) have not proven to be robust enough for integration into demanding high throughput platforms.Our approach to this challenge was to investigate alternative proteases that may be more compatible with automated on-line digestion, peptide separation, and MS detection. Pepsin, which is acid-compatible (i.e. it acts in the stomach to initially aid in the digestion of food) (13), is a particularly promising candidate. This protease has previously been successfully used for the targeted analyses of protein complexes, hydrogen/deuterium exchange experiments (14, 15), and characterization of biopharmaceuticals (16, 17). Generally, pepsin preferentially cleaves the peptide bond located on the N-terminal side of hydrophobic amino acids, such as leucine and phenylalanine, although with less specificity than the preferential cleavage observed for trypsin at arginine and lysine. The compatibility of pepsin with typical LC-MS operation makes it an ideal choice for the development of novel approaches combining protein digestion, protein/peptide separation, and MS-based protein/peptide identification.To develop an automated system capable of simultaneously capturing top-down and bottom-up data, enzyme kinetics of the chosen protease must be extremely fast (because one cannot wait hours as is typical when performing off-line proteolysis). Another requirement is the use of immobilized enzyme or a low enough concentration of the enzyme such that autolysis products do not obscure the detection of substrate peptides. The latter was a concern when using pepsin because prior hydrogen/deuterium exchange experiments used enzyme:substrate ratios up to 1:2 (18, 19). To test whether or not such a large concentration of pepsin was necessary, we performed pepsin digestion at ratios of 1:20. Many alternative energy inputs into the system were considered for speeding up the digestion. For instance, it has been shown that an input of ultrasonic energy could accelerate the reaction rate of a typical trypsin digestion while using small amounts of a protease (20). Because ultrasonic energy results in an increase of temperature and microenvironments of high pressure, it has been hypothesized that the higher temperature was the component responsible for the enhanced enzyme activity (21). López-Ferrer et al. (22, 23), however, have demonstrated that application of higher pressure with incorporation of a Barocycler alone can make trypsin display faster enzyme kinetics. This phenomenon can easily be integrated with an LC separation (which already operates at elevated pressure) to enable an automatable ultrarapid on-line digestion LC-MS proteomics platform. Herein, we refer to this platform as the fast on-line digestion system (FOLDS) (23). Although FOLDS has been described before using trypsin, here the system is characterized with pepsin, and the results obtained are compared with results attainable with trypsin. Like trypsin, pepsin produced efficient protein digestion in just a few minutes when placed under pressure. Because of the natural maximal activity of pepsin at low pH, the FOLDS can be incorporated with a RePlay (Advion Biosciences, Ithaca, NY) system, and this powerful combination is what ultimately makes the integration of top-down and bottom-up proteomics analyses possible. The integrated analysis begins with a chromatographic separation of intact proteins. The separated proteins are then split into two streams. One stream proceeds directly to the mass spectrometer for MS and/or tandem MS analysis. The second stream is split into a long capillary where the chromatographic separation of the proteins is maintained, but their arrival to the mass spectrometer for detection is delayed. This is in essence the concept of RePlay (24, 25). Herein, we have taken the RePlay a step further by implementing our FOLDS technology into the second split delayed stream of proteins. While these delayed proteins travel down the long and narrow capillary, we exposed them to pepsin where, in combination with the pressure, the proteins are quickly and reproducibly digested. These peptide fragments are subsequently subjected to MS and/or tandem MS analysis. The FOLDS RePlay system allows the rapid and robust incorporation of the integrated top-down bottom-up proteomics work flow with the ability to not only identify proteins but also to sequence multisite/combinatorial PTMs because all detected peptides (from the FOLDS analysis) are confined to the original chromatographic peak of the protein they were derived from. The analysis of protein mixtures using this integrated strategy reduces the total amount of samples required to obtain both the top-down and bottom-up data, increases throughput, and improves protein sequence coverage.     

Apoptosis is the essential target of selective pressure against p53, whereas loss of additional p53 functions facilitates carcinoma progression

The high frequency of p53 mutation in human cancers indicates the important role of p53 in suppressing tumorigenesis. It is well established that the p53 regulates multiple, distinct cellular functions such as cell-cycle arrest and apoptosis. Despite intensive studies, little is known about which function is essential, or if multiple pathways are required, for p53-dependent tumor suppression in vivo. Using a mouse brain carcinoma model that shows high selective pressure for p53 inactivation, we found that even partially abolishing p53-dependent apoptosis by Bax inactivation was sufficient to significantly reduce the selective pressure for p53 loss. This finding is consistent with previous reports that apoptosis is the primary p53 function selected against during Eμ-myc-induced mouse lymphoma progression. However, unlike observed in the Eμ-myc-induced lymphoma model, attenuation of apoptosis is not sufficient to phenocopy the aggressive tumor progression associated with complete loss of p53 activity. We conclude that apoptosis is the primary tumor suppressive p53 function and the ablation of additional p53 pleiotropic effects further exacerbates tumor progression.     

Autosomal recessive dilated cardiomyopathy due to DOLK mutations results from abnormal dystroglycan O-mannosylation

Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5-13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations.