Shell protein degradation by TMT- LC-MS/MS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Spondylus Gaederopus
TISSUE(S): Exoskeleton
SUBMITTER: 
Jorune Sakalauskaite
LAB HEAD: Beatrice Demarchi
PROVIDER: PXD025068 | Pride | 2021-11-25
REPOSITORIES: Pride
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Project description:This work reports a comprehensive and integrated microstructural, biochemical and proteomics study on the shell matrix of Spondylus gaederopus, the Mediterranean thorny oyster. We investigate the skeletal matrix proteins which are involved in biomineralization and compare the identified Spondylus sequences with other shell proteins, that are publicly available in databases. Using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated by different bleaching treatments. We identified six shell proteins, which also displayed features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. However, many reconstructed peptide sequences (de novo) could not be matched to any known shell proteins and we suggest that these probably represent lineage-specific sequences. The proteomic data implies that Spondylus may have evolved a distinct molecular toolkit for biomineralization. Using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated by different bleaching treatments. Six shell proteins were identified, which displayed features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. However, most of the reconstructed peptide sequences (de novo) could not be matched to any known shell proteins and probably represent lineage-specific sequences.
2020-03-31 | PXD016760 | Pride
Project description:Background: The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. A focus of this research has been the nacreous inner layer of the shell with its conspicuous arrangement of aragonite platelets, resembling in cross-section a brick-and-mortar wall. In comparison, the outer, less stable, calcitic prismatic layer has received much less attention. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. This was soon followed by the C-type lectin perlucin and the growth factor-binding perlustrin, both isolated from H. laevigata nacre, and the crystal growth-modulating AP7 and AP24, isolated from H. rufescens nacre. Mass spectrometry-based proteomics was subsequently applied to to Haliotis biomineralization research with the analysis of the H. asinina shell matrix and yielded 14 different shell-associated proteins. That study was the most comprehensive for a Haliotis species to date. Methods: The shell proteomes of nacre and prismatic layer of the marine gastropod Haliotis laevigata were analyzed combining mass spectrometry-based proteomics and next generation sequencing. Results: We identified 297 proteins from the nacreous shell layer and 350 proteins from the prismatic shell layer from the green lip abalone H. laevigata. Considering the overlap between the two sets we identified a total of 448 proteins. Fifty-one nacre proteins and 43 prismatic layer proteins were defined as major proteins based on their abundance at more than 0.2% of the total. The remaining proteins occurred at low abundance and may not play any significant role in shell fabrication. The overlap of major proteins between the two shell layers was 17, amounting to a total of 77 major proteins. Conclusions: The H. laevigata shell proteome shares moderate sequence similarity at the protein level with other gastropod, bivalve and more distantly related invertebrate biomineralising proteomes. Features conserved in H. laevigata and other molluscan shell proteomes include short repetitive sequences of low complexity predicted to lack intrinsic three-dimensional structure, and domains such as tyrosinase, chitin-binding, and carbonic anhydrase. This catalogue of H. laevigata shell proteins represents the most comprehensive for a haliotid and should support future efforts to elucidate the molecular mechanisms of shell assembly.
2018-06-18 | PXD009567 | Pride
Project description:In this project we have applied palaeoproteomics to study archaeological shell beads dating back to the beginning of the 7th mill. cal BCE and recovered from a prehistoric site in Jordan (Ba'ja). These beads were made of mollusc shells and were of unknown origin, but suspected to be made of larger bivalves such as Tridacna or Spondylus sp. The aim of the study was to analyse ancient proteins extracted from these beads in order to infer their biological origin. Tiny amounts of ancient samples were used and the extracted proteins were analysed by high resolution liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). In order to identify obtained peptides, we have created an ‘in-house’ molluscan protein database, by gathering molluscan sequences (proteins and transcriptomes), which were downloaded from publicly available databases in NCBI. Searching against this comprehensive database, we were able to identify a number of shell proteins from Tridacna sp., the giant clam shells. Our study reports the oldest molluscan protein sequences ever recovered that come from a very warm environment.
2024-11-04 | PXD041082 | Pride
Project description:The development of protein anti-degradation strategy is important for their storage at ambient conditions. Despite that it is known that biominerals, typical inorganic-organic composites, can preserve proteins at room temperature for a long time, it is unclear the extent of protein degradation under high temperatures. In this study, we examined protein remaining in the toasted abalone shell under high temperatures (200 and 300 °C) by biomineral proteomics method. Surprisingly, 24 proteins including carbonic anhydrase, hemocyanin, actin can still be identified from shells even after toasting under 300°C, not much decreased compared to that in the 200°C-treated and the native shell. However, the microstructure and composition (both mineral and organic matrix) of shells were altered significantly revealed by scanning electron microscopy, infrared spectroscopy, and X-ray diffraction. The well-preserved of proteins may be partially due to the sacrifice of mineral/organic interfaces and the formation of nanopores in the shell at high temperatures. Moreover, the extracted proteins from both groups were able to affect calcium carbonate in vitro. This study has potential implications in various fields such as protein storage at high temperatures and palaeoproteomics.
2022-07-01 | PXD030901 | Pride
Project description:The appearance of hard mineralized exoskeletons is a critical leap for animal evolution and partially lead to the explosion of diverse animals during the Cambrian, for example, molluscs. A majority of molluscs have mineralized shells to protect themselves. Despite numerous studies that have studied the remarkable mechanical properties of shells, the origin of shell formation is still elusive. Hence, this study investigated the overlooked shell proteome of chitons, which belong to polyplacophoran, Aculifera of Mollusca. By comparing the shell proteome to well-studied Conchifera groups, we inferred possible ancestral biomineralization toolkits of stem-group Mollusca. Taking advantage of the recently sequenced chiton mantle transcriptome and genome, eight core biomineralization proteins were identified by proteomics. Surprisingly, in contrast to previous thought that shell formation is convergently evolved, two important shell matrix proteins, Nacrein-like and Pif-like proteins were found to be conserved among Aculifera and Conchifera groups. Our findings identify a missed link of mineralized shell evolution in Mollusca and pose a hypothesis that stem-group molluscs have already evolved core biomineralization toolkits, which likely facilitate the formation of mineralized shells for protection that partially leads to their explosion.
2023-01-03 | PXD033490 | Pride
Project description:Palaeoproteomics analysis of mollusc shells (Modiolus modiolus, Ostrea edulis, Unio pictorum, Margaritifera margaritifera, Unio crassus, Pseudunio auricularius) and shell ornaments (double-buttons) from Neolithic European sites (Havnø, Hornstaad-Hörnle IA, Peştera Ungurească).
2019-05-08 | PXD011985 | Pride
Project description:Shells of pearl oyster are natural biominerals with remarkable properties and can be repaired after damage, which are regulated by biomacromolcules especially shell matrix proteins (SMPs). Identifying SMPs is critical for further understanding the process. Although proteomic methods have been used to reveal the complex protein mixture in mature shells, the proteomics of repaired shells after shell damage have not been reported before. Here, we studied the SMPs of the repaired shells (prismatic layers) 5-10 days after shell damage in Pinctada fucata by integrating transcriptomics and proteomics and compared the microstructure difference between repaired and mature shells. Although the repaired shells are calcite, similar to mature shells, the microstructure of repaired shells has holes in the center of prisms with irregular and curved edges. In total, we found 49 SMPs from the repaired shells including some proteins only existing in mature nacreous layers. Peroxidase-like protein and beta-N-acetylhexosaminidase may be important players in repaired shells. In addition, they have the capability to affect CaCO3 crystallization process in vitro, altering the packing and reducing the crystallinity of crystals. This study could improve our understanding of shell repair process and lay the foundation for studying SMPs-controlled biomineralization.
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Project description:The Manila clam (Ruditapes philippinarum) is a cultured bivalve species with high worldwide commercial importance. Nevertheless, diseases can cause high economical losses. For this reason, the study of immune genes in bivalve mollusks has increased in the last years. The present work describes the construction of the first R. philippinarum microarray containing immune-related hemocyte sequences and its application for the study of the gene transcription profiles of hemocytes from clams challenged with Vibrio alginolyticus through a time course. A comparative analysis of gene expression was conducted between R. philippinarum infected and non-infected by V. alginolyticus clam hemocytes. Clams (n=100) were notched in the shell next to the adductor muscles and injected with 100 µl of Vibrio alginolyticus, strain TA15, (10^8 UFC/ml in PBS) to mimic an intramuscular infection. Controls (n=100) were injected with 100 µl of PBS. After stimulation, clams were returned to the tanks and maintained at 15ºC until sampling at 3, 8, 24, and 72 hours after challenge Hemolymph (1 ml) was withdrawn from the adductor muscle of the clams with a 0.5mm diameter (25G) disposable needle. Hemolymph from four individuals was pooled and biological replicates were taken at each sampling point. Hemolymph was centrifuged at 4°C at 3000 g for 10 minutes. The pellet was resuspended in 250 µl of Trizol (Invitrogen). Total RNA isolation was conducted following the manufacturer's specifications in combination with the RNeasy mini kit (Qiagen) for RNA purification after DNase I treatment. Gene expression profiling was performed using an R. philippinarum oligo-DNA microarray of 13,671 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.
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