Proteomic Analysis of OPTN-Interacting Proteins

For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, proteins were separated by 10% SDS-PAGE under reducing conditions. Proteins were visualised by silver staining with ProteoSilver Plus (Sigma), bands were excised from both the OPTN-IP and control-IP gel lanes and processed for in-gel digestion and LC-MS/MS with the LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Loughborough, UK), as previously described (Mulvey et al., 2013 (link)). Raw MS files were analysed by the Mascot search engine 2.3.02 (Matrix Science, London, UK) and searched against a SwissProt human database 2013_10 (containing 39,696 entries including common contaminants). Mascot search analysis parameters included: trypsin enzyme specificity, allowance for 2 missed cleavages, peptide mass tolerance of 20 ppm for precursor ions and fragment mass tolerance of 0.8 Da. Oxidation (M) was selected as a variable modification and carbamidomethyl (C) was selected as a fixed modification.

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Chew T.S., O'Shea N.R., Sewell G.W., Oehlers S.H., Mulvey C.M., Crosier P.S., Godovac-Zimmermann J., Bloom S.L., Smith A.M, & Segal A.W. (2015). Optineurin deficiency in mice contributes to impaired cytokine secretion and neutrophil recruitment in bacteria-driven colitis. Disease Models & Mechanisms, 8(8), 817-829.

Publication 2015

ProteoSilver Plus LTQ-Orbitrap mass spectrometer Mascot search engine 2.3.02

Chromatography Cleavages Enzyme Human Ions Liquid chromatography Peptide Proteins Sds page Tandem mass spectrometry Tolerance Trypsin

Corresponding Organization :

Other organizations : University College London, University of Auckland, University of Cambridge, University College Hospital, Eastman Dental Hospital

Top 5 similar protocols

Variable analysis

independent variables

Protein separation method (10% SDS-PAGE under reducing conditions)
Protein visualization method (silver staining with ProteoSilver Plus)

dependent variables

Proteins identified by LC-MS/MS analysis
Protein abundances in OPTN-IP and control-IP samples

control variables

Trypsin enzyme specificity
Allowance for 2 missed cleavages
Peptide mass tolerance of 20 ppm for precursor ions
Fragment mass tolerance of 0.8 Da
Oxidation (M) as a variable modification
Carbamidomethyl (C) as a fixed modification

controls

Control-IP sample (negative control)

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