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J Biomol Tech. 2007 February; 18(1): 34.
PMCID: PMC2291812

P99-T A Simple Method to Quantitate Selenomethionine Incorporation into Proteins

Abstract

Selenomethionine substitution is the preferred method for preparing heavy-atom derivatives of proteins for crystal structure determination using the multi-wavelength anomalous diffraction phasing method. This approach allows researchers to take advantage of the anomalous signal from a number of diverse atoms. We recently published a protocol describing a number of variables that play a role in determining incorporation efficiency of selenomethionine into mammalian expression systems.1 Here we describe, in detail, a simple method for assessing selenomethionine substitution by replacement of methionine in homogeneous protein preparations. Using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) technology following trypsin proteolysis of the recombinant protein, we are able to evaluate variables that play roles in affecting selenomethionine incorporation. Examples will illustrate (a) the ease of identification of modified peptides containing the selenomethione and (b) relative quantitation of such peptides when compared with the control, unmodified peptides.

Notes

Key to Abstract Numbering
Prefixes: P, Poster; RG, Research Group; SP, Scientific Session Presenter; EP, Educational Session Presenter.
Following the hyphen is the designated presentation day: S, Sunday; M, Monday; T, Tuesday.

REFERENCE

1. Barton WA, Tzvetkova-Robev D, Erdjument-Bromage H, Tempst P, Nikolov DB. Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells. Protein Science 2006;15:2008–2013. [PubMed]

Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities