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BioID mass spectrometry analysis of proteins proximal to human NERCLIN

ABSTRACT: To gain insight into the function of NERCLIN in human cells, we analyzed its proximal proteins by the proximity-dependent biotin identification (BioID) approach. 143B cells were transfected with fusion constructs using jetPRIME. Following 24 h of transfection, biotin was added to each plate and allowed to biotinylate the proximal proteins for the next 24 h. The plates were washed with PBS, cells were scraped, pelleted and frozen. For each sample, four biological replicates were analyzed. Liquid chromatography mass spectrometry analysis was performed on an Orbitrap Elite ETD mass spectrometer (Thermo Scientific) using the Xcalibur version 2.7.1 coupled to a Thermo Scientific nLCII nanoflow system. The mass spec analysis was done in technical duplicates. Thermo.RAW files were searched with Proteome Discoverer 1.4 (Thermo Scientific) against SEQUEST search engine. The search parameters were taken from. All reported data were based on high confidence peptides assigned in Proteome Discoverer with a 0.01% FDR by Percolator. Control samples (GFP and AIF) and Contaminant Repository for Affinity Purification database were used to identify high confidence interactors of NERCLIN based on the spectral counts.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Markku Varjosalo Svetlana Konovalova

PROVIDER: MSV000086786 | MassIVE | Mon Feb 01 03:27:00 GMT 2021

SECONDARY ACCESSION(S): PXD023918

REPOSITORIES: MassIVE

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Project description:The hen egg vitelline membrane is a protein layer separating the yolk content from the egg white. The integrity of egg vitelline membrane is important for both the shell egg and the egg product industries. However, its physical properties are altered during prolonged storage, depending on storage temperature. The aim of this work was to identify proteomic changes of the hen egg vitelline membrane during 28-day storage at +4°C and +20°C. Vitelline membrane samples were collected from freshly-laid eggs (n=24) and 28-day-old eggs stored at 4°C (n=24) and 20°C (n=24). They were freeze-dried and solubilized using a buffer containing SDS and beta-mercaptoethanol. For proteomic analyses, equal protein amounts of pooled samples (n=4 individual samples per pool, n=6 pools per condition) were loaded on a 10% SDS-polyacrylamide separating gel. Proteins were included and concentrated in the gel after a brief migration, and stained with Coomassie Blue. Each protein band (containing the whole protein sample) was cut into small pieces and processed for mass spectrometry analysis. Following in gel digestion with trypsin, peptides were extracted and analyzed by nanoflow liquid chromatography tandem mass spectrometry (Nano LC-MS/MS) in technical triplicate. Experiments were performed on a dual linear ion trap Fourier Transform Mass Spectrometer LTQ Orbitrap Velos Pro (Thermo Fisher Scientific) coupled to an Ultimate 3000 RSLC Ultra High Pressure Liquid Chromatographer (Thermo Fisher Scientific). Data were acquired using Xcalibur version 3.0.63 software (Thermo Fisher Scientific). MS/MS ion searches were performed using Mascot search engine (Matrix Science) via Proteome Discoverer software (ThermoFisher Scientific) against NCBIprot_gallus gallus database. Peptide and protein identifications were validated using the Peptide Prophet and Protein Prophet algorithms with a probability of 95% for proteins and 99% for peptides. Protein identifications were accepted if they contained at least two identified peptides. The search parameters included trypsin as a protease with two allowed missed cleavages and carbamidomethylcysteine, methionine oxidation and acetylation of N-term protein as variable modifications. The tolerance of the ions was set to 5 ppm for parent and 0.8 Da for fragment ion matches. After applying a protein identification filter requiring a minimum of two unique peptides, a 99% peptide identification probability, and a 95% protein identification probability, the data were normalized and quantified using the eXtracted Ion Chromatogram (XIC) method in Scaffold Quant (Proteome Software).

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BioID mass spectrometry analysis of proteins proximal to human NERCLIN

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