Identification of NEM-Modified Cysteines in ALV-A Env by Mass Spectrometry

(A) ALV-A was exposed to PMB or to NHS-Biotin in the presence or absence of sTva. Env was immunoprecipitated with a polyclonal antibody against the C-terminus of TM and biotinylated proteins were detected with streptavidin-HRP. The positions of SU and TM are indicated.

(B) The purified, deglycosylated SU used for mass spectrometry analysis was quantified by SYPRO Ruby staining. The positions of SU and the PNGase F enzyme used for glycosylation are indicated. Molecular weight standards are in the left lane.

(C) Mass spectrometry identified three peptides from SU containing NEM-modified cysteine residues (indicated by *).

(D) Vertical bars represent the cysteines in SU (two of the cysteines are sequential just before position 292) and are positioned to scale. The host range (HR) determinants 1 and 2 are indicated in light grey for reference. The NEM-modified cysteines are numbered.