A Fast and Economical Sample Preparation Protocol for Interaction Proteomics Analysis

Abstract

A simple and fast immunoprecipitation (IP) protocol is designed with the sample preparation incorporated, applicable to both low and high throughput. This new protocol combines two procedures based on magnetic beads in 96‐well plate format. Protein complexes are captured by antibodies and magnetic beads conjugated with protein A. Proteins are washed and on‐bead digested by using Single‐Pot solid‐phase sample preparation (SP3). The whole IP‐SP3 approach can be completed in one day, which is considerably faster compared to the classical approach. No major quantitative differences are found between SP3 and FASP (filter‐aided sample preparation) or a longer incubation protocol. Taken together, the IP‐SP3 protocol is a fast and economical approach easily applicable for large‐scale protein interactome analysis.

Glycine Receptor Complex Analysis Using Immunoprecipitation‐Blue Native Gel Electrophoresis‐Mass Spectrometry

“…The IP samples were run on SDS‐PAGE gel, cut into three fractions, trypsin digested, and analyzed by LC‐MS/MS. This classic approach is more labor intensive and time consuming than the recently developed FASP and SP3 methods 6,17 . However, the advantages are that it allows for quality control of the IP experiments by staining the proteins on the SDS‐PAGE gel (Figure S2, Supporting Information), while the fractionation and separate LC‐MS/MS of each fraction increases the depth of the proteomics analysis.…”

“…This classic approach is more labor intensive and time consuming than the recently developed FASP and SP3 methods. 6,17 However, the advantages are that it allows for quality control of the…”

“…Immunoprecipitation of a protein using an antibody followed by proteomics analysis is a well-established technique to reveal the individual components of protein complexes. 17 In addition to the quality of the antibody, the ability to filter contaminants is equally critical to the success of the experiment. Often, hundreds of proteins are identified in a single IP, most of them are false positives bound non-specifically to the antibody or beads causing random noise.…”

“…Immunoprecipitation of a protein using an antibody followed by proteomics analysis is a well‐established technique to reveal the individual components of protein complexes 17 . In addition to the quality of the antibody, the ability to filter contaminants is equally critical to the success of the experiment.…”

The endosomal protein sorting nexin 4 is a synaptic protein

“…DIV14-15 cortical neurons (500.000 neurons per sample) were prepared as previously described 29 . In brief, cells were washed and collected in ice cold PBS with protease inhibitor cocktail (Roche).…”