Optimizing tryptic digestions for analysis of protein:protein interactions by mass spec

Protein:protein interactions (PPIs) play a key role in regulating cellular activities including DNA replication, transcription,translation, RNA splicing, protein secretion, cell cycle control and signal transduction. A comprehensive method is needed to identify the PPIs before the significance of the protein:protein interactions can be characterized. Affinity purification−mass spectrometry (AP−MS) has become the method of choice for discovering PPIs under native conditions. This method uses affinity purification of proteins under native conditions to preserve PPIs. Using this method, the protein complexes are captured by antibodies specific for the bait proteins or for tags that were introduced on the bait proteins and pulled down onto immobilized protein A/G beads. The complexes are further digested into peptides with trypsin. The protein interactors of the bait proteins are identified by quantification of the tryptic peptides via mass spectrometry.

The success of AP-MS depends on the efficiency of trypsin digestion and the recovery of the tryptic peptides for MS analysis. Several different protocols have been used for trypsin digestion of protein complexes in AP-MS studies, but no systematic studies have been conducted on the impact of trypsin digestion conditions on the identification of PPIs.  A recent publication used NFB/RelA and BRD4 as bait proteins and five different trypsin digestion conditions (two using “on beads” and three using “elution” digestion protocols). Although the performance of the trypsin digestion protocols changed slightly depending on the different bait proteins, antibodies and cell lines used, the authors of the paper found that elution digestion methods consistently outperformed on-beads digestion methods.

Reference

Zhang, Y. et al. (2017) Quantitative Assessment of the Effects of Trypsin Digestion Methods on Affinity Purification−Mass Spectrometry-based Protein−Protein Interaction Analysis
J of Proteome. Res. 16, 3068–82.

Optimized His-Tag Protein Purification from Cell-Free Expression Reactions

4453MAHis-tag is the most commonly used protein fusion tag. Current available nickel purification systems are of limited use for purifying His-tagged proteins expressed in rabbit reticulocyte lysate because of the copurification of hemoglobin present in rabbit reticulocyte lysate. This often limits the downstream applications of purified proteins in fluorescence-based functional assays or protein:protein interaction studies and also decreases the amount of proteins purified.

The MagZ™ Protein Purification System, is a unique system designed for the purification of expressed His-tagged protein from rabbit reticulocyte lysate. This protein purification system has the advantage over nickel-based immobilized metal ion affinity chromatography (IMAC) because it eliminates 99.9% of hemoglobin contamination. Continue reading