Project description:The role of stem cells in solid tumors remains controversial. In colorectal cancers (CRC), this is complicated by the conflicting ‘top-down’ or ‘bottom-up’ hypothesis of cancer initiation. We profiled the expressions of genes from the top (T) and bottom (B) fractions of the crypt in morphologically normal-appearing colonic mucosa (M) and contrasted this to that of matched mucosa adjacent to tumors (MT) in twenty three sporadic CRC patients. In thirteen patients, the genetic distance (M-MT) between the B fractions is smaller than the distance between the T fractions indicating that the expressions of significant genes diverge further in the top fractions (B<T). In the remaining ten patients, the reverse is observed (B>T). Taking genetic divergence as an intermediate endpoint, the data indicates that it is equally likely that CRC initiates from ‘top-down’ via dedifferentiated colonocytes or ‘bottom-up’ via dysregulated intestinal stem cells. This has important ramification for subsequent therapeutic considerations. Overall design: Total RNA from 23 sporadic CRC patients matched mucosa (M) and mucosa next to tumor (MT) top and bottom crypt fractions were profiled with Affymetrix GeneChip human gene 1.0 ST array. The genetic distance between M and MT (M-MT) for 80 significantly differentially expressed genes were calculated for both top and bottom fractions. The genetic distance (M-MT) for the top (T) fractions were compared to the bottom (B) fractions for every patient to decide whether the divergence is greater in the bottom fraction (B >T) or top (B<T).
Project description:Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptional responses of Pseudomonas aeruginosa growing in biofilms by using microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNAs for these housekeeping genes were low in cells at the bottom of the biofilms. Selective GFP labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNAs levels for genes regulated by the hypoxia induced regulator, Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-termanoxia. Transcripts for ribosomal proteins were primarily associated with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (rmf and PA4463) at the bottom of the biofilms. A P. aeruginosa ∆rmf strain had increased uptake of the membrane integrity stain, propidium iodide. Using selective GFP labeling and cell sorting, we showed that the dividing cells were more susceptible to tobramycin and ciprofloxacin than the dormant subpopulation. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state. 52-hour Pseudomonas aeruginosa TSA colony biofilms were cryoembedded, thin sectioned, and laser dissected (LCM) to obtain samples from the top and bottom 50 µm of the biofilms. 9 sections per biofilm were pooled. RNA was extracted with the RNeasy Micro kit, Turbo DNase treated, poly(A) tailed, and amplified using the Quantitect WTA kit. After clean up, the resulting product was fragmented and end labeled before hybridization.
Project description:Protein posttranslational methylation and acetylations have been reported to occur in archaea, including members of the genus Sulfolobus, but have not been characterized on a proteome-wide scale. Sulfolobus chromatin proteins are known to be methylated and acetylated on lysine side chains, resembling eukaryotic histones in this respect. We utilized bottom-up and top-down proteomic approaches to perform a global and deep methylation study in the hyperthermoacidophylic archaeon S. islandicus with particular interest in chromatin proteins. Without specific enrichment, 731 protein were found by bottom-up proteomic analysis. The methylation sites on >400 proteins were monitored throughout 3 cell culture growth stages: early exponential, late exponential and stationary. (The previously described aKMT4 is found to be a plausible methyltransferase responsible for the massive methylation.) The proteome-wide top-down study/approach revealed 3778 proteoforms of 681 proteins, including 292 methylated proteoforms, of which 85 were comprehensively characterized by high-resolution MS/MS. Methylated proteoforms of the five chromatin proteins were characterized in detail by combination of bottom-up and top-down data showing the differences between the closely related Sul7d proteins. The two Alba chromatin proteins are, for the first time, reported to be methylated in this work. Alba1 protein is shown to be mono-, di- and trimethylated at the lysine-16 side chain. Stage-wise top-down analysis shows that the relative abundance of the methylated proteoforms versus non-methylated ones significantly grows/increases for Alba1 and Cren7 chromatin proteins throughout the cell growth. These findings highlight the ubiquitous lysine methylation throughout the S. islandicus proteome and singificantly enrich our knowledge related aboutarchaeal chromatin proteins.
Project description:This bottom-up analysis is the supplementary data file to the top-down analysis deposited with identifier PXD014660. The peptide data are used to support assignments of modifications detected in top-down. Details are described in the associated manuscript.
Project description:Top-down and bottom-up protein analysis of venom and saliva of solenodon. Venom and saliva was separated by HPLC and either directly analysis by HR FT MS/MS (Top-down) or further decomplexed by SDS-PAGE followed by in-gel trypsine digestion and HR LC-MS/MS analysis (bottom-up, Venom only). For shotgun bottom-up comparison of venom and saliva proteins, samples were directly reduced, alkylated, digested with trypsin and measured by HPLC-MS/MS. Additional bottom-up analysis was performed from bioactivity guided fractionation experiments.
Project description:Analysis of fluorescent assembly B-phycoerythrin with a combination of bottom-up and top-down mass spectrometry to reveal heterogeneity of proteoforms and characterize their chromophorylations.
Project description:Protein posttranslational methylation and acetylation have been reported to occur in archaea, including the genus Sulfolobus, but have never been characterized on a proteome-wide scale. Among important Sulfolobus proteins carrying such modifications are the chromatin proteins that have been described to be methylated and acetylated on lysine side chains, resembling eukaryotic histones in that aspect. To get more insight into the extent of these modifications and their dynamics during the different growth steps of the thermoacidophylic archaeon S. islandicus , we performed a global and deep proteomics analysis using a combination of high-throughput bottom-up and top-down proteomics approaches on a single high-resolution mass spectrometer. 1,931 methylation sites on 751 proteins were found by the bottom-up analysis, with methylation sites of 424-526 proteins monitored throughout three cell culture growth stages: Early, Mid and Late. The previously described aKMT4 methyltransferase was found to be potentially responsible for this massive modification.
Project description:MALDI imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1,400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. This work presents a generic method and near complete list of MALDI IMS biomarkers that will become a valuable resource for the IMS community. Detailed description of the bioinformatics pipeline can be found in pipeline.txt. Briefly, different Mascot Distiller, Mascot and Scaffold versions have been used for the bottom-up and top-down data.