Computational protocol: RACK1 Associates with Muscarinic Receptors and Regulates M2 Receptor Trafficking

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Protocol publication

[…] Protein complexes were labeled and identified using a protocol developed at the Institute for Systems Biology . Samples were digested with 2 µg trypsin (Promega, sequencing grade modified, 1∶20 w/w) overnight at 37° C, then reduced with 2 µl of 250 mM Tris(2-carboxyethyl)phosphine for 30 minutes. Reduced samples were added in the dark to UV-cleavable isotope conjugated beads (d0/d6-gamma-aminobutyric acid beads ). Samples collected from mock transfected cells labeled with the light (d0) isotope and those collected from Flag-M2 transfected cells labeled with the heavy (d6) isotope. After shaking 15 minutes, the labeling reaction was quenched with 100 µl water and 2 µl beta-mercaptoethanol. d0 and d6 beads were combined and loaded onto a Bio-Spin column then washed with 1 ml each three times with 1.5 M NaCl, 6 times with 0.1% trifluoroacetic acid, 6 times with 80% N-phenylethanamide, 0.1% trifluoroacetic acid, 3 times with MeOH, 3 times with 1∶9 NH4OH:MeOH, 6 times with MeOH and 6 times with water. The beads were resuspended in 100 µl 20 mM Tris, pH 8 and 2 µl beta-mercaptoethanol. Proteins were cleaved from the beads by exposure to UV light for 2 hours and then washed from the beads 3 times with 100 µl 80% N-phenylethanamide, 0.1% trifluoroacetic acid. The sample was concentrated by Speed-Vac and resuspended in 5 µl 0.2% acetic acid . Peptides were analyzed by µHPLC-MS and data dependent µHPLC-MS/MS acquisition, selecting from each survey scan the top-three most abundant precursor ions for collision induced dissociation with a dynamic exclusion of 1 . For this, a linear ion trap mass spectrometer (LTQ; Thermo Finnigan, San Jose, CA) was used with an in-house fabricated microelectrospray source and an HP1100 solvent delivery system (Agilent, Palo Alto, CA). Samples were automatically delivered by a FAMOS autosampler (LC Packings, San Francisco, CA) to a 100 µm internal diameter fused silica capillary pre-column packed with 2 cm of 200 Å pore-size Magic C18AQ™ material (Michrom Bioresources, Auburn, CA) as described elsewhere . SEQUEST™ (Thermo Finnigan) was used to determine peptide sequence and PeptideProphet™ was used to verify correctness of peptide assignments. […]

Pipeline specifications

Software tools Comet, PeptideProphet
Application MS-based untargeted proteomics
Chemicals Acetylcholine, Carbachol