Dataset Information

Identifying Cilary Proteins in Bovine Retinas using a Gentile Extraction Method

ABSTRACT: Mutations in retinal primary cilia are responsible for human blindness but the mechanisms are not fully understood. Characterizing the proteome of an organelle such as cilia, is a fruitful way to understand its function but methods often require considerable sample quantities. Here we develop a method to isolate the primary cilia of photoreceptor cells from bovine retinas. Through LC/MS/MS proteomics analysis we identify proteins enriched for cilia function including ciliopathy disease. This study shows our method can be used to isolate retinal primary cilia to obtain sufficient quantities of native protein samples.

INSTRUMENT(S):

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Photoreceptor Cell, Retina

SUBMITTER: Kevin Drew

LAB HEAD: Kevin Drew

PROVIDER: PXD051952 | Pride | 2024-07-11

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20230207_MSC1158_bovine_retina..comet.pep.xml	Pepxml
	20230207_MSC1158_bovine_retina.MSGFplus.mzid	Mzid
	20230207_MSC1158_bovine_retina.mzXML	Mzxml
	20230207_MSC1158_bovine_retina.pep_count_mFDRpsm001	Other
	20230207_MSC1158_bovine_retina.pep_count_mFDRpsm001.log	Other

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Publications

Identifying Ciliary Proteins in Mammalian Retinas using a Gentle Extraction Method.

Fredrick Adeline S AS Claussen Erin R ER Fischer Samantha N SN Balasanyants Samson S Rajaraman Akshaya A Rosner Andi C AC Drew Kevin K

microPublication biology 20240613

Mutations in retinal primary cilia are responsible for human blindness but the mechanisms are not fully understood (Wheway et al., 2014). Characterizing the proteome of an organelle such as cilia, is a fruitful way to understand its function but methods often require considerable sample quantities. Here we develop a method to isolate the primary cilia of photoreceptor cells from bovine retinas. Through LC/MS/MS proteomics analysis we identify proteins enriched for cilia function including ciliop ...[more]

PMID: 38947246

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Project description:The outermost layer of eukaryotic cilia is a coat of membrane anchored, glycosylated proteins often referred to as glycocalyx. This highly heterogeneous layer provides functions from regulation of adhesion, force transduction and protection to signaling. We describe the structure of the ciliary coat of the green alga Chlamydomonas by cryo-electron tomography and proteomic approaches and present the high-resolution single particle structure of FMG1B via cryo-electron microscopy, the most abundant constituent of the ciliary coat. We report FMG1B to be a highly unusual mucin orthologue, which lacks the major O-glycosylation of mammalian mucins, but undergoes significant N-glycosylation. We find that an isoform of FMG1-B, FMG1-A, previously believed to be not expressed, is present in Chlamydomonas and differentially regulated with FMG1-B. By micro-flow-based adhesion assays, we observe increased surface adhesion in the glycocalyx deficient double-mutant fmg1a-fmg1b. We find this mutant to be fully capable of surface-gliding, suggesting that neither isoform is required for extracellular force transduction by intraflagellar transport. Our data provide in-depth structural details of the ciliary coat that functions as the primary contact to the environment and reveal that FMG1 acts primarily as a protective layer with adhesion-regulative properties. Data deposited here include label free quantification data on whole cells or isolated cilia confirming single knockout of FMG1B and high abundance of FMG1A in CC4533fmg1b (combined_protein_WC_CC4533fmg1b.tsv ) and double knockout of FMG1A and FMG1B in M32fmg1afmg1b (combined_proteinCilia_M32_M32fmg1afmg1b.tsv). The latter dataset was further analyzed for abundance changes in the cilia proteome (Log2_LFQ_ratios_M32_M32fmg1afmg1b.csv). Further, isolated cilia of wild type M32 were analyzed under non-reducing conditions to confirm structure predicted disulfide bridges via LC-MS/MS in FMG1B (FMG1B_intraprotein_disulfides.xlsx). Finally, isolated cilia of M32 were subjected to N-glycopeptide analyses using InSource-CID (FMG1B_HexNAc_modified_peptides.xlsx).