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J. Biol. Chem., Vol. 281, Issue 48, 37045-37056, December 1, 2006

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Analysis of Human Phagocyte Flavocytochrome b₅₅₈ by Mass Spectrometry^*

Ross M. Taylor¹, Danas Baniulis, James B. Burritt, Jeannie M. Gripentrog, Connie I. Lord, Marcia H. Riesselman, Walid S. Maaty, Brian P. Bothner, Thomas E. Angel, Edward A. Dratz, Gilda F. Linton^¶, Harry L. Malech^¶, and Algirdas J. Jesaitis

From the Departments of Microbiology and Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717 and the ^¶Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892

The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91^phox subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22^phox subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91^phox subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91^phox subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating posttranslational modifications involved in the regulation of superoxide production.

Received for publication, August 2, 2006 , and in revised form, September 8, 2006.

^* This work was supported by American Heart Association Scientist Development Grants 0630253N (to R. M. T.) and 30156 (to J. B. B.) and National Institutes of Health (NIH) Grants R01 AI 64107 (to E. A. D.), R01 GM 62547 (to E. A. D.), and RO1 AI 26711 (to A. J. J). The Montana State University Mass Spectrometry Facility is supported by National Science Foundation Grant MRI 0321267 (to E. A. D.) and the Murdock Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Montana State University, Dept. of Microbiology, 109 Lewis Hall, Bozeman, MT 59717. Tel.: 406-994-4593; Fax: 406-994-4926; E-mail: rosst{at}montana.edu.

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