Development of an Automated, Ultra-Rapid Bottom-Up Proteomics Workflow Utilizing Alginate-Based Hydrogels
Ontology highlight
ABSTRACT:
INSTRUMENT(S): timsTOF SCP
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Maor Arad
LAB HEAD: Leonard J.
PROVIDER: PXD052369 | Pride | 2024-11-12
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| checksum.txt | Txt | |||
| fasta.zip | Other | |||
| raw.zip | Other | |||
| searchtype.zip | Other |
Items per page: 5 1 - 4 of 4 |
Similar Datasets
Project description:Porcine mammary fatty tissues represent an abundant source of biomaterial for generation of breast-specific extracellular matrix (ECM). Here we report the extraction of total ECM proteins from pig breast fatty tissues, the fabrication of hydrogel scaffolds from the extracted ECM proteins, the structural properties of the scaffolds, and the applications of the hydrogel in human mammary epithelial cell spatial cultures for cell surface receptor expression, metabolomics characterization, acini formation, proliferation, migration between different scaffolding compartments, and in vivo tumor formation. This model system provides an additional option for studying human breast diseases such as breast cancer.
2019-11-12 | PXD010960 | Pride
Project description:This work highlights an automated method for enzyme immobilization via 4-triethoxysilylbutyraldehyde (TESB) and 11-triethoxysilylundecanal (TESU) derived silicone-based coupling agents. From the various coupling strategies we scrutinized, TESB and 4-triethoxysilylbutanoic acid (TESBA), the carboxylic acid derivative, were determined to be the most effective. The resulting immobilized enzyme particles (IEPs) displayed robustness and rapid digestion, while also implementing a more streamlined IEP washing procedure. The physical size of IEPs did not correlate to digestion efficiency, providing insights for potential cost-saving’s in enzyme utilization. Immobilization efficiency was determined to be 50.8 ± 7.7% as validated by Bradford's assay. Furthermore, we adapted the IEP procedure into an automated format with a Bravo liquid handler. This allowed for multiple enzymes, and/or coupling agents to be fabricated at once on a 96-well microplate, in a fraction of the time. The automated trypsin TESB and TESBA IEPs rivaled the classical in-gel digestion method for the analysis of Jurkat cell lysate, achieving similar protein group identifications. Moreover, pepsin IEPs favored cleavage at leucine (>50%) over phenylalanine, tyrosine, tryptophan, and methionine residues. Further, when miscleavages occurred, they tended to be at tyrosine residues, or when two aromatic residues were adjacent. The IEP method was then adapted for an in-situ immobilized enzyme microreactor (IMER) fabrication. We determined that TESBA could serve two roles by functionalizing the silica capillary's inner wall, and simultaneously acting as an enzyme coupler. The IMER digestion of bovine serum albumin (BSA), mirroring IEP digestion conditions, yielded a 33-40% primary sequence coverage per LC-MS/MS analysis in as little as 15 minutes. Overall, our findings underscore the potential of both IEP and IMER methods, paving the way for automated analysis and a reduction in enzyme waste through reuse, thereby contributing to a more cost-effective and timely study of the proteome.
2024-06-10 | PXD051564 | Pride
Project description:RAD51 protein is an evolutionarily conserved recombinase that plays a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. RAD51 inactivation by small molecules has been proposed as a strategy to impair the BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim to make cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully assayed in vitro by using myr-BRC4, a peptide derived from the fourth BRC repeat of BRCA2, being the strongest reported natural RAD51 binder. The present study applies a method to obtain a proteomic fingerprint for RAD51 inhibition by the myr-BRC4 peptide (designed for a more efficient cell entry) using a mass spectroscopy (MS) proteomic approach. We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteomic hits, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3) and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of RAD51 inhibition, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment.
2023-08-15 | PXD042696 | Pride
Project description:To explore the spatiotemporal regulation of ASC speck formation and inflammasome activation, we infected primary WT and Asc–/– bone marrow-derived macrophages (BMDMs) with the bacterium Francisella novicida to induce AIM2 inflammasome activation and then performed ASC IP-MS to identify proteins that interacted with ASC. We compared the IP products between WT BMDMs and Asc–/– BMDMs, and found that many proteins specifically interacted with ASC.
2024-05-29 | PXD052497 | Pride
Project description:The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18a, Mis18b and Mis18BP1), which recruits the CENP-A specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18a, 2 Mis18b and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18a can associate with centromeres and deposit CENP-A independently of Mis18b, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.
2024-09-25 | PXD052964 | Pride
Project description:The post-translationally acylated Repeats in ToXins (RTX) leukotoxins, such as the adenylate cyclase (CyaA) or α-hemolysin (HlyA), bind β2-integrins of leukocytes, but also penetrate cells lacking these receptors. We show that the indole of conserved tryptophans within the acylated segments, e.g. W876 in CyaA and W579 in HlyA, is crucial for β2-integrin-independent membrane penetration of toxins. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding or activities of CyaA W876L/F/Y toxin variants on CR3-expressing cells. In contrast, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β2-integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C. In addition, the hydrogen-deuterium exchange revealed structural changes in the acylated segment of the CyaA W876F variant, compared to intact CyaA. The substitution of W876 with a polar glutamine (W876Q), not increasing the Tm, or combining of the W876F substitution with a cavity-filling V822M substitution, decreasing the Tm of CyaA W876F+V822M to a value closer to that of CyaA, yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA was selectively impaired on erythrocytes when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. These results suggest that the bulky indole of the W876 of CyaA, or W579 of HlyA, rules the local structural flexibility of the acylated segment. This may facilitate adoption of a membrane-penetrating conformation of the toxins in the absence of β2-integrin-mediated positioning of the toxin towards the cell membrane.
2023-07-11 | PXD041207 | Pride
Project description:Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally-distinct classes of chaperones promote de novo folding, suggesting that their activity is coordinated at the ribosome. Here we use biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We find that chaperone binding is disfavoured close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor subsequently recognises compact folding intermediates exposing extensive non-native surface and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates, and recruits DnaK to solvent-accessible sites in a sequence non-specific manner. Neither Trigger factor, DnaJ nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to create a protected space for protein maturation that extends well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.
2024-07-03 | PXD048638 | Pride
Project description:Expressional alterations and post translational modifications (PTM) of type II collagen can be major cause behind osteo and rheumatoid arthritis. PTM of type II collagen α1 chain (COL2A1) such as hydroxylation of proline (P), lysine and glycosylation of hydroxylysine can act as epitopes resulting COL2A1 as autoantigen in cartilage tissues. Previous study stated proline hydroxylation (Hyp) as an important PTM in type II collagen leading to dysfunctional collagen extracellular matrix assembly in vivo. Here we report for the first time peptide mass fingerprinting (PMF) identification and tandem mass spectrometry based mapping of Hyp PTM in COL2A1 from Capra hircus (C. hircus) using Mascot database. As mascot database does not contain C. hircus COL2A1 sequence information, our identification is based on the homologous COL2A1 from Bos taurus and Homo sapience (above 98 % identity). Findings include identification of new triplet Gly-F-Hyp in C. hircus COL2A1 and well known Gly-X-Y triplet with Hyp present in the X position, instead of Y position. PMF data contains lager number of Hyp in COL2A1 from C. hircus consistent with other collagen sequences. This study suggests positional alteration of Hyp/P in Gly-X-Y triplet may be used for molecular identification and characterization of type II collagen from other sources.
2019-05-06 | PXD012928 | Pride
Project description:Expressional alterations and post translational modifications (PTM) of type II collagen can be major cause behind osteo and rheumatoid arthritis. PTM of type II collagen α1 chain (COL2A1) such as hydroxylation of proline (P), lysine and glycosylation of hydroxylysine can act as epitopes resulting COL2A1 as autoantigen in cartilage tissues. Previous study stated proline hydroxylation (Hyp) as an important PTM in type II collagen leading to dysfunctional collagen extracellular matrix assembly in vivo. Here we report for the first time peptide mass fingerprinting (PMF) identification and tandem mass spectrometry based mapping of Hyp PTM in COL2A1 from Capra hircus (C. hircus) using Mascot database. As mascot database does not contain C. hircus COL2A1 sequence information, our identification is based on the homologous COL2A1 from Bos taurus and Homo sapience (above 98 % identity). Findings include identification of new triplet Gly-F-Hyp in C. hircus COL2A1 and well known Gly-X-Y triplet with Hyp present in the X position, instead of Y position. PMF data contains lager number of Hyp in COL2A1 from C. hircus consistent with other collagen sequences. This study suggests positional alteration of Hyp/P in Gly-X-Y triplet may be used for molecular identification and characterization of type II collagen from other sources.
2019-05-06 | PXD012911 | Pride
Project description:Wistar rats, male, treated with 500 mg/Kg/day of diacetyl by gavage, during 4 weeks
2018-04-18 | PXD004504 | Pride