[Show abstract][Hide abstract] ABSTRACT: The current advances in mass spectrometry technology have led to the possibility of analyzing more complex biological samples such as entire proteomes. Here, we describe a new and powerful methodology that combines the use of the metalloendopeptidase Lys-N and strong cation exchange with mass spectrometric analysis. The approach described here allows one to separate peptides with different functional groups. The peptides we are able to isolate are N-terminal peptides, phosphorylated peptides with a single lysine, peptides with a single basic residue (lysine), and peptides with multiply basic residues. When this separation strategy is combined with tandem mass spectrometry that involves both collision-induced dissociation and electron transfer dissociation, one can achieve an optimal targeted strategy for proteome analysis.
No preview · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Specific enrichment of Mllt10 and Dot1l, and H3K79 di-/tri-methylation at c-Myc locus in mouse crypts. (A) Schematic representation of the mouse amplicons scanned in ChIP experiments by qPCR. Purified crypt and villus fractions from mouse intestine were subjected to ChIP using antibodies directed against Tcf4 (B), β-catenin (C), Mllt10/Af10 (D), Dot1l (E), di-methyl H3K79 (F), and tri-methyl H3K79 (G). Chromatin was immunoprecipitated with the specified antibodies followed by qPCR using primer pairs spanning the c-Myc locus as indicated in (A). Results are presented as percent immunoprecipitated over input and are representative of three independent experiments.
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[Show abstract][Hide abstract] ABSTRACT: Depletion of tcf7l2, mllt10/af10 and dot1l rescues mis-expression of axin2 in apcmcr/mcr zebrafish, placing these genes downstream of Apc as Wnt target gene activators (A–N). Representative whole mount in situ hybridizations for axin2 in wild type and apcmcr/mcr mutant embryos at 80 hpf injected with (A,B) buffer alone, (C,D) MO against tcf7l2, (E–H) two independent mllt10/af10 MOs, (I–L) two independent dot1l MO, and (M,N) control MO. All MOs have been coinjected with a MO against p53. All images were captured using the same exposure and represent at least three independent experiments. In parentheses number of embryos showing described phenotype per number of total embryos analyzed.
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[Show abstract][Hide abstract] ABSTRACT: Peptides identified and coverage of (A) Mllt10 and (B) Dot1l in Tcf4 complex in mouse small intestinal crypt. Amino acid sequences of Mllt10 and Dot1l detected in the Tcf4 immunoprecipitate from crypt lysates are underlined.
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[Show abstract][Hide abstract] ABSTRACT: β-catenin recruits Mllt10/Af10 to Wnt targets. (A) Ls174T CRC lysates were immunoprecipitated with antibodies against TCF4 (left panel) and β-catenin (right panel) and analyzed by Western blotting with the indicated antibodies for binding to MLLT10/Af10 and DOT1L. (B–H) β-catenin-dependent H3K79 methylation and recruitment of MLLT10 and DOT1L to c-MYC gene in Ls174T CRC. (B) Schematic representation of human c-MYC locus and amplicons scanned in ChIP assays by qPCR. ChIP experiments in Ls174T CRC uninduced or induced with Dox using antibodies against (C) TCF4, (D) β-catenin, (E) MLLT10, (F) DOT1L, (G) H3K79 dimethyl, and (H) H3K79 trimethyl. Immunoprecipitated DNA was analyzed by qPCR using primer pairs specific for the c-MYC locus as indicated. Results are presented as percent immunoprecipitated over input and are representative of three independent experiments.
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[Show abstract][Hide abstract] ABSTRACT: Mllt10/Af10 interacts directly with β-catenin. (A) Recombinant GST-fused TCF4 and β-catenin proteins were used in pull-down assays with in vitro translated S35 labeled MLLT10 to examine direct interaction. (B) Schematic representation of N-Terminal and C-Terminal MLLT10 deletion mutants and S35 labeled ΔC- and C-Terminal β-catenin deletion mutants used in GST pulldown assays (C). N-Terminal MLLT10 interacts directly with the β-catenin C-terminal domain.
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[Show abstract][Hide abstract] ABSTRACT: mRNA expression of tcf7l2, mllt10, and dot1l during zebrafish embryonic development and their depletion by splice-inhibiting MO sequences. (A) Phylogenetic tree analysis places zebrafish (Danio rerio) Mllt10 close to mouse and human sequences. (B–C) Amino acid sequence alignment of the Leucine zipper and PHD finger domains show high conservation of Mllt10 between species. (D) RT-PCR analysis of tcf7l2, mllt10, dot1l, and tbp RNA expression levels in whole embryos at different stages of embryonic development. (E) Representation of the splice-blocking MO sequences (blue) generated to deplete mRNA levels for tcf7l2, mllt10, and dot1l.
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[Show abstract][Hide abstract] ABSTRACT: Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/β-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/β-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and β-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/β-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a β-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published β-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.
[Show abstract][Hide abstract] ABSTRACT: (A) Significant overlap between differentially regulated genes in response to MLLT10 or DOT1L depletion in HEK293T cells after Wnt stimulation. Comparison of the corresponding expression pattern after siRNA suppression of MLLT10/AF10 or DOT1L in Wnt induced condition. Heatmap showing 1,116 transcripts after siRNA depletion of MLLT10 and 9 h Wnt stimulation in HEK293T cells with greater than 1.5-fold variation. Also shown is comparison of the corresponding expression pattern of genes induced after 9 h Wnt treatment and after siRNA suppression of DOT1L. Red, upregulated after MLLT10 suppression; green, downregulated after MLLT10 suppression; grey, missing data. (B) Venn diagram comparatively depicting genes suppressed upon MLLT10 and DOT1L depletion in HEK293T cells under Wnt-induced conditions.
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[Show abstract][Hide abstract] ABSTRACT: Depletion of tcf7l2, mllt10, or dot1l does not affect the expression levels of other tcf/lef family members in zebrafish embryos. RT-PCR analysis of lef1, tcf7, tcf7l1a, tcf7l1b, and tbp RNA expression levels in whole embryos injected with MO against p53 alone, or MO against p53 coinjected with MOs against tcf7l2, mllt10, dot1l, or control MO, respectively.
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[Show abstract][Hide abstract] ABSTRACT: The largest component of the human heart, the left ventricle (LV), plays a major role in delivering blood throughout the body. Therefore, an in-depth detailed quantitative proteome analysis of the human LV is a valuable resource. For this purpose, a multifaceted proteomics approach combining differential sample fractionations (gel, strong cation exchange (SCX)), enzymatic digestions (trypsin, chymotrypsin, LysN), and peptide fragmentation techniques (CID and ETcaD) was used to enhance protein sequence coverage, identification confidence and quantitative abundance determination. Using stringent criteria, 3584 distinct proteins could be identified from the latest well-annotated Swissprot database (23,000 entries). Commutatively, the over 130,000 identified MS/MS spectra were used to assess concentrations of each identified LV protein through a combination of spectral counting methods. Among the most concentrated proteins, many currently used biomarkers for detection of myocardial infarction reside. These cardiac leakage markers have a good diagnostic power, but their prognostic potential seems limited. Discovery of markers that represent etiological determinants of cardiac disease require a shift of focus towards the signaling proteome. Therefore, a protein-class centered quantitative analysis of kinases, phosphatases and GTPases was adopted. These comparative analyses revealed many cardiac involved kinases (PKA, CaMKII, ERK) to reside among the most abundant signaling proteins, and also to mediate many observed in vivo phosphorylation sites. The abundance chart of signaling proteins may assist in identifying novel functional pathways, for instance through the abundant, but relatively little known, kinases STK38L and OXSR1. The obtained quantitative protein library of the human left ventricle is a valuable resource to isolate signaling based, putative biomarkers with concentrations likely to be detectable in plasma.
Full-text · Article · Oct 2010 · Molecular BioSystems
[Show abstract][Hide abstract] ABSTRACT: Trypsin, the most widely used enzyme in proteomics, has a few caveats as it does not perform well under certain harsh sample handling conditions and creates relatively short peptides less amenable to, for instance, electron transfer dissociation. There is, thus, room for improvement using alternative proteases. Here, we evaluate the performance of such an alternative protease, the metalloendopeptidase Lys-N, in sample preparation for proteomic analyses under various experimental conditions. The experimental parameters we evaluated were protein-to-protease ratio, incubation time, temperature, and several concentrations of denaturing modifiers often used in proteomics sample handling. Our data reveal that Lys-N is still very efficient under some very harsh (denaturing) conditions (e.g., 8 M urea, 80% acetonitrile) and at temperatures as low as 4 degrees C and up to 80 degrees C but severely hampered by guanidine hydrochloride and methanol. These rather unique features make Lys-N a good candidate for a variety of applications, such as membrane proteomics and possibly H/D exchange mass spectrometry. Additionally, we show that Lys-N is capable of, in contrast to trypsin or Lys-C, cleaving adjacent to mono- and dimethylated lysines, making it a good candidate for targeted epigenetic analysis of for instance histones.
Full-text · Article · Aug 2010 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Recently, we introduced a novel proteomics method employing a metalloendopeptidase with Lys-N specificity to produce proteolytic peptides. Fragmentation spectra generated by electron transfer dissociation, for a large proportion of the Lys-N proteolytic peptides, were found to be dominated by extensive series of c-type ions. Taking advantage of this unique spectral property, we developed an algorithm, LysNDeNovo, to facilitate de novo sequencing of these peptides. LysNDeNovo contains simple and naive heuristics to demonstrate a proof of concept, i.e. that Lys-N peptide electron transfer dissociation spectra are perfectly suited for de novo interpretation. A stringent "golden" dataset of peptides identified by conventional database search algorithms was taken to validate the performance of LysNDeNovo. The results on this dataset indicate that LysNDeNovo was able to confidently identify a considerable proportion (42%), without requiring any prior genome or protein sequences. Results of similar quantity and quality could also be obtained on a much more extensive experimental dataset, illustrating the potential for higher throughput de novo sequencing using these methods.
[Show abstract][Hide abstract] ABSTRACT: Wnt signalling maintains the undifferentiated state of intestinal crypt/progenitor cells through the TCF4/beta-catenin-activating transcriptional complex. In colorectal cancer, activating mutations in Wnt pathway components lead to inappropriate activation of the TCF4/beta-catenin transcriptional programme and tumourigenesis. The mechanisms by which TCF4/beta-catenin activate key target genes are not well understood. Using a proteomics approach, we identified Tnik, a member of the germinal centre kinase family as a Tcf4 interactor in the proliferative crypts of mouse small intestine. Tnik is recruited to promoters of Wnt target genes in mouse crypts and in Ls174T colorectal cancer cells in a beta-catenin-dependent manner. Depletion of TNIK and expression of TNIK kinase mutants abrogated TCF-LEF transcription, highlighting the essential function of the kinase activity in Wnt target gene activation. In vitro binding and kinase assays show that TNIK directly binds both TCF4 and beta-catenin and phosphorylates TCF4. siRNA depletion of TNIK followed by expression array analysis showed that TNIK is an essential, specific activator of Wnt transcriptional programme. This kinase may present an attractive candidate for drug targeting in colorectal cancer.