DOI: 10.25345/c58z11
title: MassIVE MSV000086693 - TMT10 quantitative analysis of proteins interacting with Fzd1-APEX2 in live tissue proximity labeling
title: Olfactory epithelia and olfactory bulb tissue of the Fzd1::APEX2 animals and their littermate control animals were dissected in ice-cold oxygenated ACSF. The dissected tissue was cut into 1mm^3 blocks. Blocks from 1 animal were washed 3 times with ACSF, incubated in 1mL ACSF with 0.5mM biotin-phenol and 1uL Benzonase for 30mins at 37C. Then 10uL 100mM H2O2 were added. The labeling was let to proceed at RT for 1min. The reaction was stopped by adding 1mL APEX2 stop solution, 5mM sodium azide, 2mM Trolox, 1mM sodium ascorbate in 0.1M PBS. The tissue was then washed 3X with APEX2 stop solution. The tissue was next lysed with a plastic tissue pestle in OSN lysis buffer 2, containing 50mM Tris HCl, 150mM NaCl, 1% IGEPAL-CA630, 1mM EGTA, 1.5mM MgCl2, 2% FC-14, 10% SDS, 250U Benzonase, pH7.4 at RT for 1min, then at 95C for 5min. High capacity neutroavidin beads were used to enrich the biotinylated proteins from the lysate. For lysate from 1 animal, 50uL of beads were used. The beads were washed 2X with RIPA buffer before use. The denatured lysate was added to the beads and incubated at 4C overnight in the presence of 1X proteinase inhibitor, 1mM PMSF, 250U Benzonase. The beads were then washed 2X with RIPA buffer, 1X with 1M KCl, 1X with Na2CO3, 1X with 2M urea in 10mM Tris-HCl, pH8.0, 2X with RIPA buffer. After washing, beads used for each animal were incubated in 100uL elution buffer, containing 5%SDS, 0.2%FC-14, 20mM DTT, 25mM biotin, 30mM Tris-HCl, pH6.8 at 60C for 2hr, then 95C 5min. Digestion and tandem mass tag labeling of biotin-captured protein samples The precipitated proteins were dissolved in 100mM Tris-HCl, pH8.5, 8M urea, reduced in 5mM TCEP, incubated at RT for 30min, and carboxymethylated by 10mM CAM at RT for 30min in dark, digested with endoproteinase Lys-C and trypsin, at 37C ON. Digested peptides were cleaned up by SPE (Sep-Pak, Waters). Peptide concentration was measured using Thermo Scientific Pierce Quantitative Colorimetric Peptide Assay (Thermo Scientific, San Jose, CA). Then 200mM TEAB was added to a final concentration of 100mM. Based on assay results, the same amount of peptides were collected from all 11 APEX2 samples and mixed with 0.4ul of iRT standard peptides (Biogenesis, Zurich, Switzerland). Each of TMT11plex reagents (0.8mg, Thermo Fisher Scientific, Orckford, IL) was resuspended into 41ul of anhydrous acetonitrile and added to each of the 11 peptides/iRT mixtures. After 1h, the reaction was quenched by adding 8ul of 50% hydroxylamine. Differentially TMY-labeled peptides were mixed at a 1:1:1:1:1:1:1:1:1:1:1 ratio and dried down. Multiplexed mass spectrometry analysis of TMT-labeled peptide mixtures TMT-labeled peptides were analyzed on an Orbitrap Fusion Lumos MS (Thermo Scientific, San Jose, CA) coupled to a Dionex UltiMate 3000 RSCLnano System. Peptides were loaded on an Acclaim PepMap 100 C18 0.3mm i.D. 5mm length trap cartridge (Thermo Scientific, San Jose, CA). A 50um i.d. column was packed in-house with 150mm of 1.9um ReproSil-Pur C18-AQ resin (Dr. Masch GmbH, Germany). AgileSLEEVE (Analytical Sales & Products, Pompton Plains, NJ) was used to maintain column temperature to 40C. The mobile phases were water/acetonitrile/formic acid at 95:5:0.1 (v/v/v) for buffer A (pH2.6) and at 20:80:0.1 (v/v/v) for buffer B. The chromatography gradient was a 50min 2% B; a 10min ramp to 10%B; 120min from 10 to 40%B; 15min to 95%B; 15min at 95%B; 1min to 2%B; followed by a 19min in 2%B. The nano pump flow rate was set to 150nL/min. Orbitrap Fusion Lumos was set up with SPS-MS3 method as: full MS1 resolution 120,000; ITMS2 isolation window 0.7m/z, ITMS2 CID 35%; FTMS3 isolation window 0.7 m/z, FTMS3 max ion injection time 105ms, FTMS3 resolution 50,000, FTMS3 HCD energy 65%. Proteomic data analysis The LC/MS dataset was analyzed using Proteome Discoverer (version 2.4, Thermo Scientific, San Jose, CA) and R. MS/MS spectra were searched against a mouse protein database (downloaded from NCBI 2018-06-27) complemented with common contaminants. SEQUEST implemented through PD was set up as: MS1 mass tolerance 10ppm, fragment mass tolerance 0.6Dalton, up to two missed cleavage sites, static modification of C (+57.021), dynamic modification included K and peptide N-termini with TMT tag (+229.163), biotin-phenol modification of Y (+361.146), biotin modification of K (+226.0776), oxidized biotin-phenol modification of Y (+377.141), and oxidation of M (+15.995). Results were filtered to a 1% FDR at peptides levels using Percolator through PD. Proteins were quantified by summing reporter ion intensities across all matching PSMs. Difference in protein levels were assessed using ANOVA in R. The enriched proteins were uploaded to STRING database search portal for functional analysis including GO term analysis, KEGG pathway analysis, Reactome analysis, and quantitative literature search analysis. Proteins with known associations were plotted in R.