A Quantitative, High-Throughput Screen for Protein Stability

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-thro...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2000-07, Vol.97 (15), p.8296-8301
Main Authors: Ghaemmaghami, S., Fitzgerald, M. C., Oas, T. G.
Format: Article
Language:English
Subjects:
Amides
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Bacteriophage lambda - chemistry
Biochemistry
Biological Sciences
Carrier Proteins - chemistry
Carrier Proteins - genetics
Carrier Proteins - metabolism
Deuterons
DNA-Binding Proteins
Exchange rates
Hydrogen
Ligands
Maltose - genetics
Maltose - metabolism
Maltose-Binding Proteins
Mass spectra
Peptides
Plasma stability
Protein stability
Proteins
Protons
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Repressor Proteins - chemistry
Repressor Proteins - genetics
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
Viral Proteins - chemistry
Viral Proteins - genetics
Viral Regulatory and Accessory Proteins
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Summary:In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric λ repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.140111397