Efficient IP sample preparation to study protein-protein interactions

In 2.5 hours from sample collection to analysis


The function and activity of a protein are often modulated by other proteins with which it interacts. Characterizing protein–protein interactions is critical to understand protein function and the biology of the cell. Quantitative affinity purification followed by mass spectrometry is a powerful approach to investigate physical interaction partners of a protein of interest. Recent advances in technology now enable a quantification of up to 100 interactomes per day.

GFP-Traps® (ChromoTek GmbH) are ideal for efficient, one-step isolation of fluorescent fusion proteins and their interacting partners. In combination with the in-StageTip (iST) Sample Preparation Kit (PreOmics GmbH) both ensure an ideal sample preparation with high reproducibility and efficiency. The GFP-Trap® is a monovalent matrix with GFP-binding proteins coupled to it. They are suited for efficient, one-step isolation of fluorescent fusion proteins and their interacting partners. The GFP-Traps® are available either coupled to agarose beads or magnetic beads. The in-StageTip method is able to perform sample processing, from cell lysis through elution of purified peptides, in a single, enclosed volume. This robust and scalable method largely eliminates contamination or loss.1

We tested PreOmics’ iST kit and were amazed by the results. The number of identified peptides & proteins was 20–30% higher compared to what is achieved using our standard lab procedure. On top of that, the iST kit and protocol are so convenient as it includes everything ready-to-use and allows you to save a lot of time. Using the iST kit enables you to start processing your samples in the morning and have them ready for LC-MS in the afternoon. — Dr. Manuel Tzouros (project leader), Discovery Proteomics, Roche Innovation Center Basel about Preomics

Simple, fast, reproducible sample prep for mass spec

The subsequent lyse, digestion and purification is performed by the sensitive iST sample preparation kit (PreOmics GmbH), designed to assist you achieving best results with few sample preparation steps and little hands-on time. Current protocols need more than 40 hours total time and 4.5 hours hands-on-time. The iST sample preparation kit enables a robust and reproducible sample preparation with a significant time advantage compared to its competitors, reducing the hours of work down to 2.5 hours total and 1 hour hands-on-time. Mild washing buffers from both IP and iST preserve PTM-mediated protein-protein interactions therefore facilitating reproducible sample processing.

Both, trap and kit, have successfully been used in publications. Users of the iST sample preparation kit report a higher number of identified peptides and proteins – it performed better than standard methods in terms of both identification and quantification rates. Grassl et al. described their experimental workflow from saliva connection to final data analysis takes in only a total of 4 hours (with just 75 minutes for iST sample preparation) 2

“Using state-of-the-art shotgun proteomics technology, in particular the recently described iST preparation kit ... we here developed a streamlined and robust workflow for shotgun plasma proteomics. Sample preparation steps are minimized without loss of performance, and the procedure can be performed in 96-well format by a liquid handling platform. In this way, hundreds of plasma proteomes can be processed and sample preparation is not a limiting step for plasma.”3


References:

  1. Kulak NA (2014): “Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.” Nat Methods, Mar;11(3):319-24. [PMID: 24487582]
  2. Grassl N. et al (2016): "Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome”. Genome Med., 8: 44.[PMID: 4841045]
  3. Geyer PE. et al (2016): “Plasma Proteome Profiling to Assess Human Health and Disease. ”.Cell Syst., 23;2(3):185-95. [PMID: 27135364]