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RationalePurification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI). MethodsA protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed inEscherichia coliwere immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis. ResultsSmall proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarifiedE. colicell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected. ConclusionsThe immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.
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Tandem Affinity Purification (TAP) of Low-Abundance Protein Complexes in Filamentous Fungi Demonstrated Using Magnaporthe oryzae
Protein–protein interactions underlie cellular structure and function. In recent years, a number of methods have been developed for the identification of protein complexes and component proteins involved in the control of various biological pathways. Tandem affinity purification (TAP) coupled with mass spectrometry (MS) is a powerful method enabling the isolation of high-purity native protein complexes under mild conditions by performing two sequential purification steps using two different epitope tags. In this protocol, we describe a TAP-MS methodology for identifying protein-protein interactions present at very low levels in the fungal cell. Using the 6xHis-3xFLAG double tag, we start the affinity purification process for our protein of interest using high-capacity Ni²⁺ columns. This allows for greatly increased sample input compared to antibody-based first-step purification in conventional TAP protocols and provides a large amount of highly concentrated and preliminarily purified protein complexes to be used in a second purification step involving FLAG immunoprecipitation. The second step greatly facilitates the capture of low-level interacting partners under in vivo conditions. Our TAP-MS method has been proven to secure the characterization of low-abundance protein complexes under physiological conditions with high efficiency, specificity, and economy in the filamentous fungus Magnaporthe oryzae and might benefit gene function and proteomics studies in plants and other research fields.
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- Award ID(s):
- 1758805
- PAR ID:
- 10322595
- Editor(s):
- Jacob, S.
- Date Published:
- Journal Name:
- Methods in molecular biology
- Volume:
- 2356
- ISSN:
- 1940-6029
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1007/978-1-0716-1613-0_8
