Project description:The optimal conditions and procedures for efficient and reproducible protein extraction of FFPE tissues have not yet been standardised and new sensitive techniques are continually being developed and improved upon. To our knowledge, there is no general agreement as to the choice of detergent or buffer system (and/or addition of PEG 20,000) required for efficient and reproducible protein extraction from human FFPE tissues. Moreover, the effect of PEG 20,000 on protein extraction efficiency has not been evaluated using human FFPE colorectal cancer tissues. This study therefore aims to assess the impact of PEG 20,000 on the protein extraction efficiency, reproducibility, and protein selection bias of the protein extraction buffer used for FFPE colonic resection tissue in label-free LC-MS/MS analysis. The sample pellets were also tested for residual protein, not extracted in the initial extraction. The results show that the absence of PEG 20,000 increases the number of peptides and proteins identified by unfractionated LC-MS/MS analysis, and the method is more reproducible. However, no significant differences were observed with regard to protein selection bias. We propose that studies generating high protein yields would benefit from the absence of PEG 20,000 in the protein extraction buffer.
Project description:Recently, we elucidated T. urticae’s repertoire of secreted salivary proteins, revealing several members of expanded protein families with unknown functions [PMID: 27703040]. In this study, mite salivary secretions were additionally examined using a peptidomics approach.
Project description:The saliva from Bemisia tabaci (MED biotype) adults was collected using an artificial feeding system and analyzed using an LC-MS/MS proteomics analysis.
Project description:The extracellular matrix is an important part of the cellular microenvironment regulating various cellular functions. Decellularized matrices present a valuable strategy for recreating the cellular niche in vitro and implementing matrix signaling in cell culture models. However, the tissue-specificity of decellularized matrices is little considered in most in vitro models. Here, we present the generation of a porcine-derived pancreas-specific decellularized extracellular matrix scaffold (d-PanMa) and show the impact of scaffold-specific cues on stem cell culture. The proteomic analysis in this work compare the proteome of porcine pancreas (PanMa), intestine (SISser) and lung (lungMa) both in its native and decellularized form. The analysis verify the enrichment of important matrisome proteins in the decellular matrices and provide a detailed map of the protein composition and abundance across the matrices.
Project description:Protein modification by Ubiquitin or Ubiquitin-like modifiers is mediated by an enzyme cascade composed of E1s, E2s and E3s enzymes. E1, or ubiquitin-activating enzymes, perform the ubiquitin activation. Next, ubiquitin is transferred to ubiquitin-conjugating enzymes or E2s. Finally, ubiquitin ligases or E3s catalyze the transfer of ubiquitin to the acceptor proteins. E3 enzymes are responsible of determining the substrate specificity. Determining which E3 enzyme maps to which substrate is a major challenge that is greatly facilitated by the TULIP2 methodology. TULIP2 methodology is fast, precise and cost-effective. Compared to previous TULIP methodology protocol, TULIP2 methodology achieves a more than 50-fold improvement in the purification yield and two orders of magnitude improvement in the signal-to-background ratio after label free quantification by mass spectrometry analysis. The method includes the generation of TULIP2 cell lines, subsequent purification of TULIP2 conjugates, preparation and analysis of samples by mass spectrometry
Project description:Fungal proteomics is a developing field that requires renewed interest and attention from the scientific community in many aspects. One of the most compelling objectives of fungal biology is to find out how fungal pathogens colonize a host, in order to find new ways of impeding this colonization, and proteomics is an important and useful tool to this end. However, fungi are also taxonomically interesting and are important biochemical model organisms for the study of many cellular processes, such as cytoskeletal regulation. Additionally, fungi are sources of secondary metabolites, many of which serve as medications for humans. Nevertheless, much work remains in developing systems-biology approaches to understanding fungal gene expression and secondary metabolism. Current fungal proteomics approaches involve 2D SDS-PAGE and extensive, complex, protein extraction methodologies. In this work, an application of a modified Folch extraction to protein extraction was used to perform de novo peptide sequencing of the proteome from the plant and human pathogen Lasiodiplodia theobromae, which greatly streamlined and simplified the analysis process. Using a metaproteomics bioinformatics approach, many novel proteins for L. theobromae were identified and targeted for further biochemical characterization and annotation efforts.
Project description:Extracellular vesicles was isolated from plasma of healthy and psychotic patients. Changes in proteomes was assessed on individual samples (n=68) by LC-MS/MS.
Project description:We performed microarray analyses on human ESCs-derived NPs and neurons carrying loss-of-function mutation in the MeCP2 gene. Neural precursors and differentiating neurons at 2 and 4-week were used for RNA extraction and affymetrix microarrays. We added ERCC RNA spike-in controls to normalize to cell number input.
Project description:We aimed to compare CTCF binding patterns, chromatin states and 3D genome structure in the absence and after activation of Wnt signaling in HEK293T cells.