Analysis of human phagocyte flavocytochrome b(558) by mass spectrometry.
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.
Taylor
Ross M
RM
Departments of Microbiology and Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, USA. rosst@montana.edu
Baniulis
Danas
D
Burritt
James B
JB
Gripentrog
Jeannie M
JM
Lord
Connie I
CI
Riesselman
Marcia H
MH
Maaty
Walid S
WS
Bothner
Brian P
BP
Angel
Thomas E
TE
Dratz
Edward A
EA
Linton
Gilda F
GF
Malech
Harry L
HL
Jesaitis
Algirdas J
AJ
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
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