Analysis of human phagocyte flavocytochrome b(558) by mass spectrometry

• Published in: The Journal of biological chemistry Vol. 281; no. 48; p. 37045
• Main Authors: Taylor, Ross M, Baniulis, Danas, Burritt, James B, Gripentrog, Jeannie M, Lord, Connie I, Riesselman, Marcia H, Maaty, Walid S, Bothner, Brian P, Angel, Thomas E, Dratz, Edward A, Linton, Gilda F, Malech, Harry L, Jesaitis, Algirdas J
• Format: Journal Article
• Language: English
• Published: United States 01-12-2006
• Subjects: Amino Acid Sequence; Chryseobacterium - metabolism; Cytochrome b Group - chemistry; Cytochrome b Group - physiology; Glycosylation; Humans; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - chemistry; Membrane Glycoproteins - metabolism; Molecular Sequence Data; NADPH Oxidase 2; NADPH Oxidases - chemistry; NADPH Oxidases - metabolism; NADPH Oxidases - physiology; Neutrophils - metabolism; Phagocytosis; Recombinant Proteins - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Superoxides - metabolism; Trypsin - chemistry
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M607354200

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.