Journal of Mass Spectrometry 2013-02-01

Direct differentiation of A-ring single attachment versus A- and D-ring double attachment of phycoerythrobilin chromophores to phycobiliproteins using MALDI mass spectrometry.

M Nazim Boutaghou, Christina M Kronfel, Leanora S Hernandez, Avijit Biswas, Wendy M Schluchter, Richard B Cole

Index: J. Mass Spectrom. 48(2) , 187-92, (2013)

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Abstract

Bilin chromophore attachment to phycobiliproteins is an enzyme-catalyzed post-translational modification process. Bilin-lyases attach a bilin chromophore to their cognate protein through a thioether bond between the chromophore and a cysteine moiety. Bilin chromophores are attached to their phycobiliproteins through the 3(1) carbon of the bilin. Double attachment may also occur, and in this case, carbons 3(1) and 18(1) of the bilin are both forming covalent linkages to cysteine moieties. There is a mass spectrometric limitation when examining tryptic peptides containing two (or more) cysteines if one seeks to ascertain whether chromopeptides are singly or doubly attached. The problem is that singly and doubly attached chromopeptides appear at the same m/z value; thus, up until the present, only NMR analysis has been successful at determining whether the chromophore is singly or doubly attached. We report in this work a new, fast and accurate method for discriminating singly from doubly attached chromophores using MALDI-TOF mass spectrometry. This method was developed from mass spectral analysis of chromopeptides that had undergone in vitro or in vivo attachment of bilin chromophores to phycobiliproteins. Distinction is based on a characteristic neutral loss that appears in the MALDI-TOF mass spectrum only when the bilin is singly attached.Copyright © 2013 John Wiley & Sons, Ltd.