BioID :

BioID is a method to screen for physiologically relevant protein interactions that occur in living cells. This technique uses a promiscuous biotin ligase (Bir A) to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation.

1) Sample Preperation (Tissue lysis and Trypsinization):

Will require cell pellet or animal tissue. Approximatly 5-10 mg of proteins are extracted from the samples using the Guanidine based protein extraction method. Recovered proteins are denatured in 8M urea buffer and digested with trypsin. Peptides are then purified using solid phase extraction and quantified using BCA assay for Biotin enrichment.

 2) Biotin Enrichment using Streptadivin Beads:

We will use Streptadivin beads to bind the biotin labeled lysine residues of trypsin digested protein which has been shown to have markedly improved ability to enrich and detect endogenous biotinylated peptides by mass spectrometry (MS).

3) LC-MS Analysis:

 The enriched peptides are run on a optimized 2 hour LC-MS run using a 25 cm C18 (BCH 1.7 micron) column using HCD fragmentation. Additional ETD fragmentation can be requested for additional charges. ETD fragmentation can increase the number of Biotinylated sites detected in the samples.