Tandem Affinity Purification (TAP)-MS Service

What is Tandem Affinity Purification (TAP)-MS

Tandem affinity purification (TAP) is a rapid technique used to study protein-protein interactions in vivo. Through two steps of specific affinity purification, TAP allows for the rapid isolation of proteins that interact with the target protein under physiological conditions. Originally developed for use in yeast, the TAP method has rapidly evolved due to its versatility, efficiency, high purity, and low false positives. It has since been successfully applied to the study of protein interactions in various other organisms, including mammals and plants.

The originally designed TAP tag primarily consists of two IgG-binding domains of Staphylococcus aureus protein A (ProtA) and a calmodulin-binding peptide (CBP), separated by a cleavage site for the TEV protease.

(Lukas Alfons Huber.Nature Reviews Molecular Cell Biology·2003)

Principles of Tandem Affinity Purification (TAP)

The basic principle of Tandem Affinity Purification (TAP) involves the construction of a fusion target protein with two affinity purification tags. This fusion target protein is then transfected into host cells or tissues, where the tagged target protein is expressed and binds to relevant interacting proteins. Subsequently, the interaction is captured using the tags and corresponding magnetic beads to obtain a protein complex close to its natural condition. Then, the proteins eluted from the experimental and control groups are subjected to mass spectrometry analysis. By subtracting the proteins identified in the control group from the experimental results, the proteins interacting with the target protein can be identified.

Advantages of TAP Technology

It allows the expression of fusion target proteins in cellular physiological or near-physiological conditions, enabling the study of target proteins and their unknown associated protein complexes, thus more accurately reflecting the functions of proteins in living organisms.

With two elution steps, it reduces the amount of non-specific proteins, making it more sensitive and less prone to false positives compared to yeast two-hybrid assays.

It is suitable for large-scale protein-protein interaction studies.

It can reveal complex protein associations, not only identifying directly interacting proteins but also detecting indirectly interacting proteins and even capturing small molecules beyond proteins.

There are various types of TAP tags available, allowing researchers to choose and design tags according to their specific research needs, making the technique widely applicable and practical.

When combined with mass spectrometry, TAP technology provides high-confidence protein correlations through bioinformatics analysis, saving a significant amount of time in subsequent validation work.

TAP Experimental Workflow

Applications of TAP Technology

The combination of Tandem Affinity Purification (TAP) with mass spectrometry (TAP-MS) has become an efficient and practical method widely used in the field of biomolecular interactions. In recent years, with comprehensive improvements in TAP tags and continuous advancements in mass spectrometry technology, TAP-MS has significantly improved sensitivity and specificity. This has enabled the technique to play a crucial role in more diverse fields and made it possible for us to conduct systematic studies of protein complexes.

TAP-MS Service Contents

Construction of fusion target protein eukaryotic expression vectors or viral vectors with trep-Flag or 6*His-Flag tags.

Transient transfection of target cells with vectors or packaging virus-infected target cells.

TAP purification of the target protein complex separately using Strep-Flag or 6*His-Flag tags.

Enzymatic digestion of eluates from experimental and control groups.

LC-MS/MS analysis to obtain quantitative and qualitative information of proteins.

Subtracting proteins identified in the control group from the experimental results to identify proteins interacting with the target protein.

Technical Platform

热科学问Exactive(高分辨率质量spectrometry).

Protein Sample Requirements

Delivery

Results of protein identification from the interaction study (Excel spreadsheet).

Detailed experimental procedures, instrument used, source of reagents, software retrieval parameters, etc.

Chromosomal translocation-derived aberrant Rab22a drives metastasis of osteosarcoma

Journal: Nature Cell Biology.

Published: 2020

Abstract

Osteosarcoma is an aggressive malignant bone tumor that frequently metastasizes to the lungs, leading to poor prognosis. However, the molecular mechanisms underlying osteosarcoma lung metastasis remain largely unknown. Here, the authors identified exonic-intronic fusion genes in osteosarcoma cell lines and tissues. They discovered an interaction between Rab22a-NeoF1 and SmgGDS-607 by using theTAP-MSand demonstrated that synthetic peptides can disrupt this interaction, thereby preventing lung metastasis in an in situ model of osteosarcoma. This finding may offer a promising therapeutic strategy for osteosarcoma patients with lung metastasis.

Result

The authors' research indicated that Rab22a-NeoF1-2 is more stable compared to Rab22a-NeoF3-6, and Rab22a-NeoF1 is the main functional fusion gene, prompting the authors to focus on Rab22a-NeoF1 for further investigation. They generated a monoclonal antibody (RAD5-8) and a humanized antibody (hRAD5-8-v1-R5) against Rab22a-NeoF1. Additionally, two siRNAs targeting the intron sequences of DOK5 (the gene fused with Rab22a) were designed, which effectively knocked down the expression of RAB22A-NeoF1 without affecting the mRNA levels of rab22a or DOK5. A nanoparticle-nucleic acid complex polymer system has been developed and proven to be efficient and safe. To effectively use siRNA targeting RAB22A-NeoF1 for inhibiting osteosarcoma lung metastasis in vivo, PEG-PEI was employed to deliver RAB22A-NeoF1 siRNA. The experimental results further demonstrated that the functions of endogenous Rab22a-NeoFs in both ZOS and ZOS-m cells are primarily governed by Rab22a-NeoF1, suggesting that Rab22a-NeoF1 could be a potential therapeutic target for metastatic osteosarcoma patients.

深入研究Rab22a-NeoF1的机制promotes osteosarcoma lung metastasis, the authors utilized theTAP-MStechnique to explore proteins interacting with Rab22a-NeoF1. Consequently, they identified proteins from the RhoA family, and a series of experimental results indicated that Rab22a-NeoF1 enhances osteosarcoma lung metastasis by activating RhoA. Further mining of TAP-MS data focused on RAP1GDS1 (also known as SmgGDS and GDS1), a specific non-canonical guanine nucleotide exchange factor for RhoA and RhoC. Experimental results suggested that Rab22a-NeoF1-mediated osteosarcoma lung metastasis requires association with SmgGDS-607 (an isoform).

By precisely deciphering the interacting regions, the authors confirmed that Arg4 and Lys7 of Rab22a-NeoF1 are essential for its binding to SmgGDS-607, thereby promoting lung metastasis. To validate the therapeutic potential of this interaction, the authors used a targeted peptide based on the Rab22a(WT) sequence to interfere with the interaction between Rab22a-neof1 and SmgGDS-607. The results demonstrated that this interference could abolish Rab22a-neof1-induced lung metastasis, providing a promising therapeutic strategy for osteosarcoma patients with lung metastasis.