Protein staining, mass spectrometry compatible

Lauber WM, Carroll JA, Dufield DR, Kiesel JR, Radabaugh MR, Malone JP. Mass spectrometry compatibility of two-dimensional gel protein stains. Electrophoresis 2001 22(5) 906-918. 

Ideally, the gel should have been stained with a Coomassie stain—preferably a colloidal Coomassie if detection sensitivity is an issue, e.g., (2,3), or one of the commercially available stains. This method is also compatible with Sypro stains (though an ultraviolet transilluminator will be required to visualize the protein bands or spots during excision). Standard silver stains are not compatible, but certain modified silver stains—e.g., (4), or one of the commercially available mass spectrometry compatible silver stains—can be used however, even these usually give inferior mass spectrometry data compared to Coomassie- or Sypro-stained gels. 

Stain the gel to visualize protein samples using a mass spectrometry-compatible silver staining kit, SimplyBlue stain, or SYPRO Ruby stain (see Note 19). Follow the manufacturer s instructions for staining. 

CBB G-250 is another popular total protein stain. Researchers blotting 2-D PAGE gels particularly favor it because it is compatible with mass spectrometry. Stained blots provide good media for archiving 2-D PAGE separations. A version of SYPRO Ruby, formulated for blots, is a very sensitive total protein stain. 

Yan, J. X. Wait, R. Berkelman, T. Harry, R. A. Westbrook, J. A. Wheeler, . H. Dunn, M. J. A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 2000, 21(11), 3666-3672. 

CBB provides a simple few-step method for protein visualization and offers detection of proteins down to 10 ng per spot (depending on gel thickness). New commercial colloidal CBB stains do provide a dynamic range of detection, with good compatibility with mass spectrometry for protein identification, but even these new dyes are not linear within the order of magnitude of protein levels found in the cell. 

Following separation, proteins need to be visualized, and not all staining methods are compatible with subsequent recovery of peptide fragments after proteolytic digestion. Furthermore, to make full use of the high sensitivity of mass spectrometry, staining needs to reach detection levels in the low- to subpmol... 

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