title: Analysis of human phagocyte flavocytochrome b(558) by mass spectrometry.
abstract: 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.
authors.affiliation: Departments of Microbiology and Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, USA. rosst@montana.edu
authors.forename: Ross M
authors.initials: RM
authors.lastname: Taylor
authors.forename: Danas
authors.initials: D
authors.lastname: Baniulis
authors.forename: James B
authors.initials: JB
authors.lastname: Burritt
authors.forename: Jeannie M
authors.initials: JM
authors.lastname: Gripentrog
authors.forename: Connie I
authors.initials: CI
authors.lastname: Lord
authors.forename: Marcia H
authors.initials: MH
authors.lastname: Riesselman
authors.forename: Walid S
authors.initials: WS
authors.lastname: Maaty
authors.forename: Brian P
authors.initials: BP
authors.lastname: Bothner
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Angel
authors.forename: Edward A
authors.initials: EA
authors.lastname: Dratz
authors.forename: Gilda F
authors.initials: GF
authors.lastname: Linton
authors.forename: Harry L
authors.initials: HL
authors.lastname: Malech
authors.forename: Algirdas J
authors.initials: AJ
authors.lastname: Jesaitis
mesh_terms: D000595:Amino Acid Sequence; D045247:Chryseobacterium / Q000378:metabolism; D003573:Cytochrome b Group / Q000737:chemistry* / Q000502:physiology*; D006031:Glycosylation; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D009504:Neutrophils / Q000378:metabolism; D010587:Phagocytosis; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013481:Superoxides / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
chemical_list: D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D013481:Superoxides; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin