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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.

A heme-bearing polypeptide core of human neutrophil flavocytochrome b(558) was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochrome b that had been exposed to endoproteinase Glu-C for 1 h. The fragment was composed of two polypeptides of 60-66 and 17 kDa by SDS-polyacrylamide gel electrophoresis and retained a native heme absorbance spectrum that was stable for several days when stored at 4 degrees C in detergent-containing buffer. These properties suggested that the majority of the flavocytochrome b heme environment remained intact. Continued digestion up to 4.5 h yielded several heme-associated fragments that were variable in composition between experiments. Digestion beyond 4.5 h resulted in a gradual loss of recoverable heme. N-Linked deglycosylation and reduction and alkylation of the 1-h digestion fragment did not affect the electrophoretic mobility of the 17-kDa fragment but reduced the 60-66-kDa fragment to 39 kDa. Sequence and immunoblot analyses identified the fragments as the NH(2)-terminal 320-363 amino acid residues of gp91(phox) and the NH(2)-terminal 169-171 amino acid residues of p22(phox). These findings provide direct evidence that the primarily hydrophobic NH(2)-terminal regions of flavocytochrome b are responsible for heme ligation.