Analysis of Human Phagocyte Flavocytochrome b 558

The catalytic core of the phagocyte NADPH oxidase is a het- erodimeric 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 nanoelectros- pray 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 hydropho- bic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-trans- lational modifications, the isolated gp91 phox subunit was subject to sequential in-gel digestion with Flavobacterium meningosep- ticum 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, meth- ods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modifica- tion 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 pro- vides analytical methods that will be useful for evaluating post- translational modifications involved in the regulation of superox- ide production.