Articles Combining Fluorescence Detection and Mass Spectrometric Analysis for Comprehensive and Quantitative Analysis of Redox-Sensitive Cysteines in Native Membrane Proteins Evgeniy V. Petrotchenko, Dan Pasek, Phillip Elms, † Nikolay V. Dokholyan, Gerhard Meissner, and Christoph H. Borchers* Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599 Monobromobimane (MBB) is a lipophilic reagent that selectively modifies free cysteine residues in proteins. Because of its lipophilic character, MBB is capable of labeling cysteine residues in membrane proteins under native conditions. Reaction of MBB with the sulfhydryl groups of free cysteines leads to formation of highly fluorescent derivatives. Here we describe a procedure for the detection and relative quantitation of MBB-labeled cysteines using fluorescence and mass spectrometric analyses, which allow determination of free cysteine content and unambiguous identification of MBB-modified cysteine residues. We have applied this approach to the analysis of the free and redox-sensitive cysteine residues of a large membrane protein, the sarcoplasmic reticulum Ca 2+ release channel with a molecular mass of 2.2 million Da. Labeling was performed under physiologic conditions where the channel complex is in its native environment and is functionally active. The purified MBB-labeled channel complex was enzymatically digested, and the resulting peptides were separated by reversed-phase high- performance chromatography. MBB-labeled peptides were detected by fluorescence and identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Under MALDI conditions, partial photolytic fragmentation of the MBB-peptide bound occurred, thus allowing convenient screening for the MBB-modified peptides in the MS spectrum by detection of the specific mass increment of 190.07 Da for MBB-modified cysteine residues. Modification of the peptides was further con- firmed by tandem mass spectrometric analysis, utilizing sequencing information and the presence of the specific immonium ion for the MBB-modified cysteine residues at m/z 266.6. Quantitative information was obtained by comparison of both fluorescence and MS signal intensities of MBB-modified peptides. Combination of fluorescence with MS detection and analysis of MBB-labeled peptides supported by a customized software program provides a convenient method for identifying and quantifying redox- sensitive cysteines in membrane proteins of native bio- logical systems. Identification of one redox-sensitive cysteine (2327) in the native membrane-bound sarco- plasmic reticulum Ca 2+ release channel is described. Proteins contain free cysteines whose oxidation regulates a wide variety of cellular functions. 1-3 Thiol redox state and protein activity are dependent on O 2 tension and glutathione redox potential and are modified by reactive oxygen species such as superoxide anion (O 2 - ) or hydrogen peroxide. Reactive oxygen species, which are produced during normal cellular function, act as second messenger molecules to control a wide variety of cellular mechanisms including transcription, inflammation, and contractility, but when produced in excess impose on cells an oxidative stress that can lead to lipid peroxidation, DNA and protein damage, and finally cell death. 1-3 Determination of mechanisms involved in redox modification of cellular components has therefore become an important topic in biology and medicine. Thiol derivatization is commonly used to study the function of free thiols in proteins. Two of the best-known classes of reagents are N-maleimide and acyl halides, both of which under controlled conditions yield highly specific sulfhydryl group modifications. 4 Recently, isotopically labeled, thiol-specific reagents have been introduced to derivatize free thiols. This facilitates mass spectro- metric detection and identification of the labeled cysteine residues in proteins. 5 The use of isotopically labeled reagents allows rapid and facile detection of labeled peptides, based on the presence of doublets in the mass spectra, corresponding to “heavy” and “light” * To whom correspondence should be addressed. Tel.: (919) 843-5310. Fax.: (919) 966-2852. E-mail: christoph_borchers@med.unc.edu. † Present address: Department of Biophysics, University of Berkeley, CA 94720-3200. (1) Matalon, S.; Hardiman, K. M.; Jain, L.; Eaton, D. C.; Kotlikoff, M.; Eu, J. P.; Sun, J.; Meissner, G.; Stamler, J. S. Am. J. Physiol. 2003, 285, L1184- L1189. (2) Soberman, R. J. J. Clin. Invest. 2003, 111, 571-574. (3) Hare, J. M.; Stamler, J. S. J. Clin. Invest. 2005, 115, 509-517. (4) Haugland, R. Handbook of fluorescent probes and research products, 9th ed.; Molecular Probes, Inc., 2002. (5) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. Anal. Chem. 2006, 78, 7959-7966 10.1021/ac060238r CCC: $33.50 © 2006 American Chemical Society Analytical Chemistry, Vol. 78, No. 23, December 1, 2006 7959 Published on Web 10/11/2006