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Shotgun proteomics as a viable approach for biological discovery in the Pacific oyster.

ABSTRACT: Shotgun proteomics offers an efficient means to characterize proteins in a complex mixture, particularly when sufficient genomic resources are available. In order to assess the practical application of shotgun proteomics in the Pacific oyster (Crassostrea gigas), liquid chromatography coupled with tandem mass spectrometry was used to characterize the gill proteome. Using information from the recently published Pacific oyster genome, 1043 proteins were identified. Biological samples (n?=?4) and corresponding technical replicates (three) were similar in both specific proteins identified and expression, as determined by normalized spectral abundance factor. A majority of the proteins identified (703) were present in all biological samples. Functional analysis of the protein repertoire illustrates that these proteins represent a wide range of biological processes, supporting the dynamic function of the gill. These insights are important for understanding environmental influences on the oyster, because the gill tissue acts as the interface between the oyster and its environment. In silico analysis indicated that this sequencing effort identified a large proportion of the complete gill proteome. Together, these data demonstrate that shotgun sequencing is a viable approach for biological discovery and will play an important role in future studies of oyster physiology.

SUBMITTER: Timmins-Schiffman E

PROVIDER: S-EPMC4732435 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Shotgun proteomics as a viable approach for biological discovery in the Pacific oyster.

Timmins-Schiffman Emma E Nunn Brook L BL Goodlett David R DR Roberts Steven B SB

Conservation physiology 20130517 1

Shotgun proteomics offers an efficient means to characterize proteins in a complex mixture, particularly when sufficient genomic resources are available. In order to assess the practical application of shotgun proteomics in the Pacific oyster (Crassostrea gigas), liquid chromatography coupled with tandem mass spectrometry was used to characterize the gill proteome. Using information from the recently published Pacific oyster genome, 1043 proteins were identified. Biological samples (n = 4) and c ...[more]

PMID: 27293593

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Project description:BackgroundBiomarkers in colorectal cancer are scarce, especially for patients with Stage 2 disease. The aim of our study was to identify potential prognostic biomarkers from colorectal cancers using a novel combination of approaches, whereby digital pathology is coupled to shotgun proteomics followed by validation of candidates by immunohistochemistry (IHC) using digital image analysis (DIA).Methods and resultsTissue cores were punched from formalin-fixed paraffin-embedded colorectal cancers from patients with Stage 2 and 3 disease (n = 26, each). Protein extraction and liquid chromatography-mass spectrometry (MS) followed by analysis using three different methods were performed. Fold changes were evaluated. The candidate biomarker was validated by IHC on a series of 413 colorectal cancers from surgically treated patients using a next-generation tissue microarray. DIA was performed by using a pan-cytokeratin serial alignment and quantifying staining within the tumor and normal tissue epithelium. Analysis was done in QuPath and Brightness_Max scores were used for statistical analysis and clinicopathological associations. MS identified 1947 proteins with at least two unique peptides. To reinforce the validity of the biomarker candidates, only proteins showing a significant (P < 0.05) fold-change using all three analysis methods were considered. Eight were identified, and of these, cathepsin B was selected for further validation. DIA revealed strong associations between higher cathepsin B expression and less aggressive tumor features, including tumor node metastasis stage and lymphatic vessel and venous vessel invasion (P < 0.001, all). Cathepsin B was associated with more favorable survival in univariate analysis only.ConclusionsOur results present a novel approach to biomarker discovery that includes MS and digital pathology. Cathepsin B expression analyzed by DIA within the tumor epithelial compartment was identified as a strong feature of less aggressive tumor behavior and favorable outcome, a finding that should be further investigated on a more functional level.

Dataset Information

Shotgun proteomics as a viable approach for biological discovery in the Pacific oyster.

Publications

Shotgun proteomics as a viable approach for biological discovery in the Pacific oyster.

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