Proteomic Analysis of Retina Samples

For proteome analysis, immunoprecipitated proteins were reduced, alkylated and trypsin digested as previously described [57 (link)]. Desalted tryptic peptides were analyzed on an Orbitrap Lumos Tribrid mass spectrometer coupled with a UltiMate 3000-nLC (Thermo Fisher Scientific). Raw data files were processed with Proteome Discoverer (v2.2, Thermo Fisher Scientific) software, using Mascot v2.5.1 (Matrix Sciences) search node for peptide/protein identification. Target Decoy was used to calculate the false discovery rate (FDR) of peptide spectrum matches, set to a p-value <0.05 [58 (link)]. Geneset enrichment analysis with fold change ratio of peptide spectral match counts between KO and WT retina was performed as described in the RNA seq analyses section below.

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Hargrove-Grimes P., Mondal A.K., Gumerson J., Nellissery J., Aponte A.M., Gieser L., Qian H., Fariss R.N., Bonifacino J.S., Li T, & Swaroop A. (2020). Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration. PLoS Genetics, 16(12), e1009259.

Publication 2020

UltiMate 3000-nLC Proteome Discoverer

Node protein Peptide Proteins Proteome Retina Rna seq Tryptic

Corresponding Organization : National Institutes of Health

Other organizations : George Washington University, National Heart Lung and Blood Institute, Eunice Kennedy Shriver National Institute of Child Health and Human Development

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Variable analysis

independent variables

Genetic knockout (KO) of a particular gene in the retina

dependent variables

Protein expression levels between KO and wild-type (WT) retina samples
Peptide spectral match counts between KO and WT retina samples

control variables

Experimental procedures for reducing, alkylating, and trypsin digesting the immunoprecipitated proteins
Desalting of tryptic peptides
Mass spectrometry analysis using an Orbitrap Lumos Tribrid mass spectrometer coupled with a UltiMate 3000-nLC
Data processing using Proteome Discoverer (v2.2) software and Mascot v2.5.1 search node
Target Decoy approach to calculate the false discovery rate (FDR) of peptide spectrum matches, set to a p-value <0.05

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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