Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels

Analytical Biochemistry
S L Cohen, B T Chait

Abstract

In this report we describe a novel approach to the mass spectrometric analysis of whole proteins from gels. The strategy consists of three components: conventional SDS-PAGE gels, reversible negative staining procedures, and passive elution of proteins from gels followed by mass spectrometric analysis. Protein bands are excised from SDS-PAGE gels, destained, and extracted. For gel loadings > or = 25 pmol of soluble protein, the proteins can be directly extracted into a solution consisting of formic acid/water/2-propanol. The recovered protein is suitable for matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization mass spectrometric analysis. For gel loadings < 25 pmol protein, the mass spectrometric response, using the direct extraction procedure, drops off sharply, an outcome that is attributed to protein recovery losses. To offset the protein losses, the extraction procedure is slightly modified by performing the passive extraction of the gel with a saturated MALDI matrix solution. During the extraction period, the matrix is allowed to crystallize, forming a suspension in solution. Protein that elutes from the gel has a chance to cocrystallize with the matrix that can be retrieved for MALDI-MS analysis. ...Continue Reading

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