Monique Slijper

Quantitative phospho-proteomics of early elicitor signalling in Arabidopsis

Perception of general elicitors by plant cells initiates signal transduction cascades that are regulated by protein phosphorylation. The earliest signaling events occur within minutes and include ion fluxes across the plasma membrane, activation of MAPKs, and the formation of reactive oxygen species. The phosphorylation events that regulate these signaling cascades are largely unknown. Here we present a mass spectrometry-based quantitative phosphoproteomics approach that identified differentially phosphorylated sites in signaling and response proteins from Arabidopsis cells treated with either flg22 or xylanase. Our approach was sensitive enough to quantitate phosphorylation on low abundance signaling proteins such as calcium-dependent protein kinases and receptor-like kinase family members. With this approach we identified one or more differentially phosphorylated sites in 76 membrane-associated proteins including a number of defense-related proteins. Our data on phosphorylation indicate a high degree of complexity at the level of post-translational modification as exemplified by the complex modification patterns of respiratory burst oxidase protein D. Furthermore the data also suggest that protein translocation and vesicle traffic are important aspects of early signaling and defense in response to general elicitors. Our study presents the largest quantitative Arabidopsis phosphoproteomics data set to date and provides a new resource that can be used to gain novel insight into plant defense signal transduction and early defense response.

Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.

The analysis of proteome-wide phosphorylation events is still a major analytical challenge because of the enormous complexity of protein phosphorylation networks. In this work, we evaluate the complementarity of Lys-N, Lys-C, and trypsin with regard to their ability to contribute to the global analysis of the phosphoproteome. A refined version of low-pH strong cation exchange was used to efficiently separate N-terminally acetylated, phosphorylated, and nonmodified peptides. A total of 5036 nonredundant phosphopeptides could be identified with a false discovery rate of <1% from 1 mg of protein using a combination of the three enzymes. Our data revealed that the overlap between the phosphopeptide data sets generated with different proteases was marginal, whereas the overlap between two similarly generated tryptic data sets was found to be at least 4 times higher. In this way, the parallel use of Lys-N and trypsin enabled a 72% increase in the number of detected phosphopeptides as compared to trypsin alone, whereas a trypsin replicate experiment only led to a 25% increase. Thus, when focusing solely on the trypsin and Lys-N data, we identified 4671 nonredundant phosphopeptides. Further analysis of the detected sites showed that the Lys-N and trypsin data sets were enriched in significantly different phosphorylation motifs, further evidencing that multiprotease approaches are very valuable in phosphoproteome analyses.

The fluxes through glycolytic enzymes in Saccharomyces cerevisiae are predominantly regulated at posttranscriptional levels

Metabolic fluxes may be regulated “hierarchically,” e.g., by changes of gene expression that adjust enzyme capacities (Vmax) and/or “metabolically” by interactions of enzymes with substrates, products, or allosteric effectors. In the present study, a method is developed to dissect the hierarchical regulation into contributions by transcription, translation, protein degradation, and posttranslational modification. The method was applied to the regulation of fluxes through individual glycolytic enzymes when the yeast Saccharomyces cerevisiae was confronted with the absence of oxygen and the presence of benzoic acid depleting its ATP. Metabolic regulation largely contributed to the ≈10-fold change in flux through the glycolytic enzymes. This contribution varied from 50 to 80%, depending on the glycolytic step and the cultivation condition tested. Within the 50–20% hierarchical regulation of fluxes, transcription played a minor role, whereas regulation of protein synthesis or degradation was the most important. These also contributed to 75–100% of the regulation of protein levels.

Full-length cytokeratin-19 is released by human tumor cells: a potential role in metastatic progression of breast cancer.

IntroductionWe evaluated whether CK19, one of the main cytoskeleton proteins of epithelial cells, is released as full-length protein from viable tumor cells and whether this property is relevant for metastatic progression in breast cancer patients.MethodsEPISPOT (EPithelial ImmunoSPOT) assays were performed to analyze the release of full-length CK19 by carcinoma cells of various origins, and the sequence of CK19 was analyzed with mass spectrometry. Additional functional experiments with cycloheximide, Brefeldin A, or vincristine were done to analyze the biology of the CK19-release. CK19-EPISPOT was used to detect disseminated tumor cells in bone marrow (BM) of 45 breast cancer patients who were then followed up over a median of 6 years.ResultsCK19 was expressed and released by colorectal (HT-29, HCT116, Caco-2) and breast (MCF-7, SKBR3, and MDA-MB-231) cancer cell lines. The CK19-EPISPOT was more sensitive than the CK19-ELISA. Dual fluorescent EPISPOT with antibodies against different CK19 epitopes showed the release of the full-length CK19, which was confirmed by mass spectrometry. Functional experiments indicated that CK19 release was an active process and not simply the consequence of cell death. CK19-releasing cells (RCs) were detectable in BM of 44% to 70% of breast cancer patients. This incidence and the number of CK19-RCs were correlated to the presence of overt metastases, and patients with CK19-RCs had a reduced survival as compared with patients without these cells (P = 0.025, log-rank test; P = 0.0019, hazard ratio, 4.7; multivariate analysis).ConclusionsFull-length CK19 is released by viable epithelial tumor cells, and CK19-RCs might constitute a biologically active subset of breast cancer cells with high metastatic properties.

Proteome analysis of yeast response to various nutrient limitations

We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and 15N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose‐repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids β‐oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the post‐transcriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology.

Toxicogenomics of bromobenzene hepatotoxicity: a combined transcriptomics and proteomics approach

Toxicogenomics is a novel approach integrating the expression analysis of thousands of genes (transcriptomics) or proteins (proteomics) with classical methods in toxicology. Effects at the molecular level are related to pathophysiological changes of the organisms, enabling detailed comparison of mechanisms and early detection and prediction of toxicity. This report addresses the value of the combined use of transcriptomics and proteomics technologies in toxicology. Acute hepatotoxicity was induced in rats by bromobenzene administration resulting in depleted glutathione levels and reduced average body weights, 24hr after dosage. These physiological symptoms coincided with many changes of hepatic mRNA and protein content. Gene induction confirmed involvement of glutathione-S-transferase isozymes and epoxide hydrolase in bromobenzene metabolism and identified many genes possibly relevant in bromobenzene toxicity. Observed glutathione depletion coincided with induction of the key enzyme in glutathione biosynthesis, gamma-glutamylcysteine synthetase. Oxidative stress was apparent from strong upregulation of heme oxygenase, peroxiredoxin 1 and other genes. Bromobenzene-induced protein degradation was suggested from two-dimensional gel electrophoresis, upregulated mRNA levels for proteasome subunits and lysosomal cathepsin L, whereas also genes were upregulated with a role in protein synthesis. Both protein and gene expression profiles from treated rats were clearly distinct from controls as shown by principal component analysis, and several proteins found to significantly change upon bromobenzene treatment were identified by mass spectrometry. A modest overlap in results from proteomics and transcriptomics was found. This work indicates that transcriptomics and proteomics technologies are complementary to each other and provide new possibilities in molecular toxicology.

Structural Characterization and Immunogenicity in Wild-Type and Immune Tolerant Mice of Degraded Recombinant Human Interferon Alpha2b

PurposeThis study was conducted to study the influence of protein structure on the immunogenicity in wild-type and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b).MethodsRhIFNα2b was degraded by metal-catalyzed oxidation (M), cross-linking with glutaraldehyde (G), oxidation with hydrogen peroxide (H), and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reverse-phase high-pressure liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. The immunogenicity of the products was evaluated in wild-type mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by enzyme-linked immunosorbent assay or surface plasmon resonance.ResultsM-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Nontreated (N) rhIFNα2b was immunogenic in the wild-type mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The anti-rhIFNα2b antibody levels in the wild-type mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ∼ N-rhIFNα2b ≫ B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance.ConclusionsRhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.

Differential Oxidation of Protein-tyrosine Phosphatases

Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs). Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTPα-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTPα-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTPα-D1 and RPTPα-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTPμ-D1, with a similar conformation as RPTPα-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H2O2 concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.

Profiling of N-Acetylated Protein Termini Provides In-depth Insights into the N-terminal Nature of the Proteome

N-terminal processing of proteins is a process affecting a large part of the eukaryotic proteome. Although N-terminal processing is an essential process, not many large inventories are available, in particular not for human proteins. Here we show that by using dedicated mass spectrometry-based proteomics techniques it is possible to unravel N-terminal processing in a semicomprehensive way. Our multiprotease approach led to the identification of 1391 acetylated human protein N termini in HEK293 cells and revealed that the role of the penultimate position on the cleavage efficiency by the methionine aminopeptidases is essentially conserved from Escherichia coli to human. Sequence analysis and comparisons of amino acid frequencies in the data sets of experimentally derived N-acetylated peptides from Drosophila melanogaster, Saccharomyces cerevisiae, and Halobacterium salinarum showed an exceptionally higher frequency of alanine residues at the penultimate position of human proteins, whereas the penultimate position in S. cerevisiae and H. salinarum is predominantly a serine. Genome-wide comparisons revealed that this effect is not related to protein N-terminal processing but can be traced back to characteristics of the genome.

Double Standards in Quantitative Proteomics Direct Comparative Assessment of Difference in Gel Electrophoresis and Metabolic Stable Isotope Labeling

Quantitative protein expression profiling is a crucial part of proteomics and requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. In this report we evaluate in a direct comparative assessment two state-of-the-art quantitative proteomic approaches, namely difference in gel electrophoresis (DiGE) and metabolic stable isotope labeling. Therefore, Saccharomyces cerevisiae was grown under well defined experimental conditions in chemostats under two single nutrient-limited growth conditions using 14N- or 15N-labeled ammonium sulfate as the single nitrogen source. Following lysis and protein extraction from the two yeast samples, the proteins were fluorescently labeled using different fluorescent CyDyes. Subsequently, the yeast samples were mixed, and the proteins were separated by two-dimensional gel electrophoresis. Following in-gel digestion, the resulting peptides were analyzed by mass spectrometry using a MALDI-TOF mass spectrometer. Relative ratios in protein expression between these two yeast samples were determined using both DiGE and metabolic stable isotope labeling. Focusing on a small, albeit representative, set of proteins covering the whole gel range, including some protein isoforms and ranging from low to high abundance, we observe that the correlation between these two methods of quantification is good with the differential ratios determined following the equation RMet.Lab. = 0.98RDiGE with r2 = 0.89. Although the correlation between DiGE and metabolic stable isotope labeling is exceptionally good, we do observe and discuss (dis)advantages of both methods as well as in relation to other (quantitative) approaches.